Posts Tagged ‘development’

Gene therapy research is booming. Since the U.S. Food and Drug Administration (FDA) issued its first approval for a gene therapyin 2017, oncology researchers have been breaking barriers in gene therapy trials, followed by an explosion in mRNA research during the COVID pandemic. Today, this trailblazing science is providing new ways to approach rare diseases and new hope when other investigational interventions have failed. In fact, themajorityof approved gene therapies are for rare diseases 14 are currently in Phase III trials for 10 rare diseases and 45 gene therapies are in early stages of development to treat 30 rare diseases. We see great potential for gene therapies, said Leslie Johnston, senior vice president of biotech delivery for Parexel. As more products are approved, it will gain traction and more companies will look to expand their therapies into other therapeutic indications. This progress presents tremendous potential to change more patients lives across many different diseases. This could be gene therapys moment. But to fully seize it, the industry must clear some complex hurdles. Gene therapies pose several unique challenges for clinical research, including ethical and safety considerations, regulatory hurdles, precarious logistics, and potentially staggering costs. These challenges may already be having ramifications: New U.S. patients treated with gene therapies approved or in development areexpected to fallby one-third from 2025 to 2034. The key to clearing these hurdles? Cooperation between sponsors, sites, regulators, patients, and other stakeholders is essential to expediting the advancement of life-saving gene therapies. Regulators should address risks without limiting innovation Gene therapy trials are strictly regulated and rightly so, due to the novel nature of the intervention and the potential long-term consequences. Gene therapy interventions also carry inherent safety risks, including the potential for unintended genetic changes or adverse immune reactions. Ensuring patient safety requires rigorous monitoring and adherence to strict protocols. However, obtaining regulatory approval under these conditions is time consuming and resource intensive. To avoid hampering scientific progress, regulators should aim to ensure that requirements are appropriately rigorous without being unmanageably onerous. Thankfully, the FDA is paying close attention to gene therapy and has demonstrated a desire to work with drug developers toward the success and approval of these treatments. Dr. Peter Marks, Director of the Center for Biologics Evaluation and Research (CBER) at the FDA, has expressed his hope for an exponential, if not logarithmic, increase in gene therapy approvals. There is a lot of excitement that this could potentially make a big difference for the treatment of human disease, said Dr. Marks in hisremarksto the National Press Forum last November. The FDA is going beyond mere rhapsodizing and taking action to accelerate gene therapy. Last year, the agencylaunched a pilot programcalled Support for Clinical Trials Advancing Rare Disease Therapeutics, or START. This program is designed to accelerate the development and approval process for treatments targeting rare diseases by providing regulatory guidance, assistance, and incentives to sponsors conducting clinical trials in this field. The program represents an important step forward in fostering innovation and collaboration between regulatory bodies and sponsors. In addition, the FDA is working toharmonize global requirementsfor the review of gene therapies. Encouraging and facilitating international cooperation and harmonization of regulatory standards including mutual recognition agreements and shared regulatory pathways for multinational clinical trials can help streamline gene therapy development globally and help bring innovations to patients faster. Even with this progress, regulators should continue to help accelerate gene therapy research by streamlining regulatory pathways specifically tailored to gene therapies. This means providing clear guidance on requirements for preclinical and clinical development, fostering collaboration between stakeholders to share knowledge and best practices, and offering expedited review processes for gene therapy products aimed at treating serious or life-threatening diseases. With a staggering2,500 cell and gene therapyinvestigational new drug applications (INDs) on file, the FDA approved justfivecell and gene therapies in 2023. Dr. Marks hassuggestedthat accelerated approval, which has successfully advanced cancer and HIV/AIDS treatments, may be the most appropriate path for this new category of treatments. But, regulators also need to commit to proactively partner with developers to understand the patient population and the risks and benefits of each new therapy. Likewise, researchers, industry stakeholders, and patient advocacy groups should engage with regulators to help them understand the unique challenges and opportunities in the field of gene therapy. This can help regulators adapt regulatory frameworks to ensure patient safety while expediting the development and approval of promising treatments. Sites and sponsors must be prepared Of course, sites and sponsors also have a crucial part to play in advancing this promising field of medicine. Clinical trial sites should enhance their capacity to conduct gene therapy trials safely and effectively and sponsors should do their part to assist sites in these efforts. By working closely with clinicians and regulators, sponsors can ensure that the trial development process aligns with clinical needs and regulatory standards. Sponsors should have a thorough understanding of FDA requirements pertaining to design, preclinical testing, and long-term follow-up. Better alignment from the outset will lead to more efficient trial designs, faster regulatory approvals, and ultimately quicker patient access to therapies. For example, sponsors working with mRNA and other genetically engineered therapies in North America not only have to go through institutional review board (IRB) review, they also have to navigate additional requirements from the U.S. National Institutes of Health (NIH) Office of Science PolicyGuidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(NIH Guidelines). These requirements usually involve an additional biosafety risk assessment review from an institutional biosafety committee (IBC) in addition to IRB review. NIH Guidelines apply for any research involving recombinant or synthetic nucleic acids (e.g. genetically engineered materials) that receives NIH support or takes place at sites that have received NIH support for such research. Even when there is zero NIH support, IBC review is considered a best practice. IBC review and inspection helps sites ensure they are fully prepared by identifying areas for improved biosafety protections and calling out gaps in current standard operating procedures (SOPs). Proactive coordination and integration of these separate review processes can speed trial timelines and help sponsors consistently address any potential concerns or issues. Sites can also be better prepared by pre-registering an IBC. The NIH takes six to eight weeks or more to approve a new registration, in addition to IBC review time so by registering an IBC before they even have a trial, sites can save a month or more in startup time over a site that waited to register. Clinical trial sites looking to host gene therapy studies must be prepared in other ways, as well, both in terms of knowledge and infrastructure. Gene therapy studies require specialized infrastructure for manufacturing, storing, and administering genetic material to adhere to strict biosafety guidelines. Something as simple as having an upholstered chair in the infusion room which would pose an unacceptable contamination risk if genetic materials were to spill would require the site to rethink their current processes. Rigorous training is also key due to the added risk of spreading genetic material to caregivers and others in close contact with patients. Research staff must be specially trained to handle, deliver, and dispose of this material safely. Of course, these measures can seem intimidating for sites that are already cost-constrained. Large academic medical centers with more resources and experience are more likely to be well-positioned for these studies. For instance, they may already have conducted bench, animal, and/or agricultural research with genetic engineering or have the funding to make any needed adjustments such as purchasing special equipment. But to maximize the potential number of sites where this research can be conducted and therefore reach more potential participants sponsors might consider providing help in the form of financial assistance, training curricula, SOP guidance, and more to smaller sites seeking to conduct gene therapy research. Logistical complexities depending on the investigational medicine and therapeutic area are among the most complicated challenges in gene therapy trials, added Johnston. From collecting the specimen from the patient, modifying it, storing it, transporting it, and then returning it back to the patient all comes with tremendously unique logistical challenges and requires equally unique equipment, technology, and expertise. And it can be cost-prohibitive. Patients must be fully on board Of course, the most essential stakeholder in any clinical trial is the patient. In gene therapy research, which can be particularly demanding, patients must have a complete understanding of and commitment to their involvement. Understanding the potential risks and benefits can help patients make informed decisions and navigate the study process. First, it's crucial for patients to adhere strictly to the protocol provided by the clinical trial team, including following medication schedules, maintaining specific hygiene practices, and attending all study visits. They should strive to maintain optimal health to enhance the body's response to gene therapy. And to avoid delays, patients should maintain open and honest communication with the clinical trial team, reporting any changes in symptoms, side effects, or general health as soon as they occur. Trial participants also need to be in it for the long haul. Because gene therapy interventions aim to produce lasting effects, even cures, they typically require long-term patient follow-up to assess efficacy and safety. But they may also need to have incredible patience. Johnston explained, There are many complexities that can impact study progress. For example, unpredictable logistical challenges like a weather event or vehicle accident could delay a temperature-sensitive delivery to a site, or data review outcomes could require an indeterminate pause period. Patience and agility are must-haves, but it is difficult for patients potentially depending on this new therapy to save or change their lives. Lastly, the industry cannot forget the patient. Involving patients and patient advocacy groups in the regulatory process can help ensure that the development of gene therapies is aligned with patient needs and priorities, as well as shed light on risk-benefit perspectives from a patients viewpoint. The more these perspectives are considered from the beginning, the greater the chance of a trials success. Rita Naman, co-founder of the Mighty Milo Foundation, emphasizes the need for a more collaborative and patient-centered approach to gene therapy development. "For ultra-rare diseases likeSPAX5, gene therapy offers a glimmer of hope where traditional treatments do not. But logistical hurdles make these therapies expensive and inaccessible, explained Naman. Closer collaboration with patients, industry, and regulators could streamline these processes, drive costs down, and speed trials. Patients like my son, and their caregivers, plus advocacy groups should be invited into the earliest discussions to prevent false starts or missed milestones in gene therapy development especially as the patients priorities dont always line up with the sponsors. In the fight for gene therapy breakthroughs, cooperation is key. The road to operationalizing gene therapy clinical trials is laced with land mines and potholes. To capture the full potential of novel gene therapy research, a new level of collaboration between sponsors, CROs, sites, oversight committees, regulatory bodies, and patients is paramount. Patients want access to novel gene treatments, and they want it fast. Sponsors want to deliver but fight logistical and financial obstacles. Regulators want to ensure safety first, especially considering such new, promising science, concluded Johnston. These three goals may seem conflicting at times, so we need to strike a balance of safety and speed, so patients dont miss their only potential treatment opportunity. A seasoned industry veteran with more than 25 years of experience, James Riddle is senior vice president of global review operations at Advarra. Riddles expertise includes large program management and growth, operational processes, development and implementation of technology solutions, and management of large Human Research Protection Programs (HRPP), Biosafety programs (IBC) and Institutional Animal Care and Use programs (IACUC). Riddle has directed numerous clients in achieving Part 11 compliance and meeting computer system validation requirements.

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Gene Therapy is Having its Moment: Can the Clinical Research Ecosystem Seize It? - Contract Pharma

The global gene therapy market size was valued at USD 8.75 billion in 2023 and is poised to grow from USD 10.47 billion in 2024 to USD 52.40 billion by 2033, growing at a CAGR of 19.6% in the forecast period (2024-2033).

Gene therapy is a technique that uses a gene to treat, prevent or cure a disease or medical disorder. Often, gene therapy works by adding new copies of a gene that is broken, or by replacing a defective or missing gene in a patients cells with a healthy version of that gene. Both inherited genetic diseases (e.g., hemophilia and sickle cell disease) and acquired disorders (e.g., leukemia) have been treated with gene therapy.

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The development of the market is owing to an increase in the number of gene therapy-based discoveries, increasing investment in this sector, and rising approval of gene therapy products. According to the WHO, 10 to 20 new cell and gene therapies are expected to be approved each year by 2025.

Continuous developments in recombinant DNA technology are anticipated to enhance the efficiency of gene therapy in the coming years. Hence, ongoing progresses in recombinant DNA technology are anticipated to expand the number of ongoing clinical trials for gene therapy. Primarily, these advancements are taking place in the context of various gene-editing tools and expression systems to augment the R&D for products. The advent of CRISPR/Cas9 nuclease, ZFN, and TALEN allows easy & precise genome editing. As a result, in recent times, the gene-editing space has witnessed a substantial number of research activities, which, in turn, is expected to influence the growth of the gene therapy market.

The growth of the gene therapy market is expected to be majorly benefitted from the increasing prevalence of cancer. The ongoing increase in cancer patients and related death per year emphasizes the essential for the development of robust treatment solutions. In 2020, there were around 18.1 million new cases of cancer worldwide. 9.3 million of these cases involved men, while 8.8 million involved women. Continuing developments in tumor genetic studies have delivered substantial information about cancer-related molecular signatures, which in turn, is expected to support ongoing clinical trials for cancer therapeutics.

With rising demand for robust disease treatment therapies, companies have focused their efforts to accelerate R&D for effective genetic therapies that target the cause of disease at a genomic level. . Furthermore, the U.S. FDA provides constant support for innovations in this sector via a number of policies with regard to product manufacturing. In January 2020, the agency released six final guidelines on the manufacturing and clinical development of safe and efficient products.

Furthermore, facility expansion for cell and gene therapies is one of the major factors driving the gene therapy market growth. Several in-house facilities and CDMOs for gene therapy manufacturing have begun investing to enhance their production capacity, which, in turn, is anticipated to create lucrative opportunities for market players. For instance, in April 2022, the FDA approved commercial licensure approval to Novartis for its Durham, N.C. site. This approval permits the 170,000 square-foot facility to make, test, and issue commercial Zolgensma, as well as manufacture therapy products for current & upcoming clinical trials.

Cell and Gene Therapy Market :https://www.biospace.com/article/releases/u-s-cell-and-gene-therapy-clinical-trial-services-industry-is-rising-rapidly/

Gene Therapy Market Report Highlights

U.S. Gene Therapy Market Size in U.S. 2024 to 2033

The U.S. gene therapy market size was estimated at USD 3.19 billion in 2023 and is projected to surpass around USD 18.50 billion by 2033 at a CAGR of 19.22 % from 2024 to 2033.

North America dominated the market in 2023 with the largest revenue share of 65.12% in 2023. This region is expected to become the largest routine manufacturer of gene therapy in terms of the number of approvals and revenue generated during the forecast period. Increasing investments in R&D from large and small companies in the development of ideal therapy drugs are anticipated to further boost the market.

Furthermore, the increasing number of investments by the governments and the growing prevalence of targeted diseases are the factors fueling the market. According to the Spinal Muscular Atrophy Foundation, in 2020, around 10,000 to 25,000 children and adults in the U.S. were affected by spinal muscular atrophy, making it a fairly common disease among rare diseases.

Europe is estimated to be the fastest-growing regional segment from 2024 to 2030. This is attributed to its large population with unmet medical needs and increasing demand for novel technologies in the treatment of rare but increasingly prevalent diseases. Asia Pacific market for commercial application of genetic therapies is anticipated to witness significant growth in the forecast period, which can be attributed to the easy availability of resources, local presence of major companies, and increased investment, by the governments.

UK Gene Therapy Market

The UK gene therapy market is anticipated to witness accelerated growth over the forecast period, due to increased investments by various big companies and governments, including the NHS & research laboratories. For instance, in March 2022, the UK government invested USD 326.45 million to accelerate healthcare research and manufacturing. Under this investment, additional $80 million of the fund will help companies at the forefront of invention with their commercial-scale manufacturing investments in areas like gene and cell therapies, as well as improved diagnostic technologies, among others. Various mergers & partnerships between manufacturers, universities, and other government bodies are expected to boost the market over the forecast period.

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What is gene therapy used for?

Most gene therapies are still in the clinical trial phase. Clinical trials play an important role in finding treatments that are safe and effective. Clinical trials are investigating gene therapy for the treatment ofcancer,macular degenerationand other eye diseases, certaingenetic conditionsandHIV/AIDS.

The U.S. Food and Drug Administration (FDA) has approved two gene therapies for use in the U.S.:

Is gene therapy safe?

The first gene therapy trial was run more than thirty years ago. The earliest studies showed that gene therapy could have very serious health risks, such as toxicity, inflammation, and cancer. Since then, researchers have studied the mechanisms and developed improved techniques that are less likely to cause dangerous immune reactions or cancer. Because gene therapy techniques are relatively new, some risks may be unpredictable; however, medical researchers, institutions, and regulatory agencies are working to ensure that gene therapy research, clinical trials, and approved treatments are as safe as possible.

Comprehensive federal laws, regulations, and guidelines help protect people who participate in research studies (called clinical trials). The U.S. Food and Drug Administration (FDA) regulates all gene therapy products in the United States and oversees research in this area. Researchers who wish to test an approach in a clinical trial must first obtain permission from the FDA. The FDA has the authority to reject or suspend clinical trials that are suspected of being unsafe for participants.

The National Institutes of Health (NIH) also plays an important role in ensuring the safety of gene therapy research. NIH provides guidelines for investigators and institutions (such as universities and hospitals) to follow when conducting clinical trials with gene therapy. These guidelines state that clinical trials at institutions receiving NIH funding for this type of research must be registered with the NIH Office of Biotechnology Activities. The protocol, or plan, for each clinical trial is then reviewed by the NIH Recombinant DNA Advisory Committee (RAC) to determine whether it raises medical, ethical, or safety issues that warrant further discussion at a RAC public meeting.

An Institutional Review Board (IRB) and an Institutional Biosafety Committee (IBC) must approve each gene therapy clinical trial before it can be carried out. An IRB is a committee of scientific and medical advisors and consumers that reviews all research within an institution. An IBC is a group that reviews and approves an institution's potentially hazardous research studies. Multiple levels of evaluation and oversight ensure that safety concerns are a top priority in the planning and carrying out of gene therapy research.

The clinical trial process occurs in three phases. Phase I studies determine if a treatment is safe for people and identify its side effects. Phase II studies determine if the treatment is effective, meaning whether it works. Phase III studies compare the new treatment to the current treatments available. Doctors want to know whether the new treatment works better or has fewer side effects than the standard treatment. The FDA reviews the results of the clinical trial. If it determines that the benefits of the new treatment outweigh the side effects, it approves the therapy, and doctors can use it to treat a disorder.

What are CAR T cell therapy, RNA therapy, and other genetic therapies?

Several treatments have been developed that involve genetic material but are typically not considered gene therapy. Some of these methods alter DNA for a slightly different use than gene therapy. Others do not alter genes themselves, but they change whether or how a genes instructions are carried out to make proteins.

Cell-based gene therapy

CAR T cell therapy (or chimeric antigen receptor T cell therapy) is an example of cell-based gene therapy. This type of treatment combines the technologies of gene therapy and cell therapy. Cell therapy introduces cells to the body that have a particular function to help treat a disease. In cell-based gene therapy, the cells have been genetically altered to give them the special function. CAR T cell therapy introduces a gene to a persons T cells, which are a type of immune cell. This gene provides instructions for making a protein, called the chimeric antigen receptor (CAR), that attaches to cancer cells. The modified immune cells can specifically attack cancer cells.

RNA therapy

Several techniques, called RNA therapies, use pieces of RNA, which is a type of genetic material similar to DNA, to help treat a disorder. In many of these techniques, the pieces of RNA interact with a molecule calledmessenger RNA(or mRNA for short). In cells, mRNA uses the information in genes to create a blueprint for making proteins. By interacting with mRNA, these therapies influence how much protein is produced from a gene, which can compensate for the effects of a genetic alteration. Examples of these RNA therapies include antisense oligonucleotide (ASO), small interfering RNA (siRNA), and microRNA (miRNA) therapies. An RNA therapy called RNA aptamer therapy introduces small pieces of RNA that attach directly to proteins to alter their function.

Epigenetic therapy

Another gene-related therapy, called epigenetic therapy, affectsepigenetic changesin cells. Epigenetic changes are specific modifications (often called tags) attached to DNA that control whether genes are turned on or off. Abnormal patterns of epigenetic modifications alter gene activity and, subsequently, protein production. Epigenetic therapies are used to correct epigenetic errors that underlie genetic disorders.

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Vector Insights

The AAV segment shows a significant revenue contribution of 22.9% in 2023. Several biopharma companies are offering their viral vector platform for the development of AAV-based gene therapy product. For instance, in September 2016, Lonza signed an exclusive agreement with Massachusetts Eye and Ear to support its novel Anc-AAV gene therapy platform for development and commercialization of next-generation gene therapies based on their AAV platform. Similarly, RegenxBio had made an agreement with companies AveXis & Biogen in March 2014 and May 2016, respectively, which would allow both companies to use RegenxBios AAV vector platform for development of gene therapy molecules. Furthermore, in May 2021, Biogen Inc. and Capsigen Inc. entered into a strategic research partnership to engineer novel AAV capsids that have the possibility to deliver transformative gene therapies, which can address the fundamental genetic causes of numerous neuromuscular and CNS disorders. In July 2021, the U.S. Department of Commerces National Institute of Standards and Technology (NIST), National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL), and United States Pharmacopeia (USP) announced a collaboration to evaluate analytical methods and develop standards for AAV. As part of this partnership, NIST and USP will be conducting an interlaboratory study in which several laboratories will measure these serious quality attributes, and their results will be linked and examined. This collaboration will support the development of new promising gene therapies that will significantly advance peoples lives.

Indication Insights

The spinal muscular atrophy (SMA) segment dominated the market in 2023. Although SMA is a rare disorder, it is one of the most common fatal inherited diseases of infancy. The development of Zolgensma (AVXS-101), has proven its effectiveness in treating SMA and altering the phenotype of the illness. The FDA approved Novartis' Zolgensma approval in May 2019, which is aimed at treating the underlying cause of SMA. As of now, Zolgensma is the only gene treatment in this field to have been approved. The approval of this gene therapy is evidence of the growing use of therapies to treat serious hereditary illnesses like SMA.

The Beta-Thalassemia Major/SCD segment is anticipated to register the fastest CAGR over the forecast period. Gene therapy for SCD and -thalassemia is based on transplantation of gene-modified hematopoietic stem cells. Clinical and preclinical studies have shown the efficacy and safety of this therapeutic modality. However, several other factors, such as suboptimal gene expression levels & gene transfer efficiency, limited stem-cell dose and quality, and toxicity of myeloablative regimens are still hampering its efficacy. Despite these challenges, in June 2019, bluebird Bios Zynteglo (formerly LentiGlobin) received conditional approval in Europe for the treatment of -thalassemia and is expected to receive U.S. FDA approval in August 2022. Moreover, the product has already received Orphan Drug status by the U.S. FDA for treatment of patients with sickle cell disease (SCD). Furthermore, in April 2021, Vertex Pharmaceuticals and CRISPR Therapeutics amended partnership for the development, production, and commercialization of CTX001 in sickle beta thalassemia and cell disease. These achievements in this segment are anticipated to significantly boost the adoption of the product in this segment.

Route of Administration Insights

The intravenous segment dominated the global gene therapy market in 2023. Large number of approved products along with strong pipeline for IV candidates is the major reason for the segment dominance. The segment is also expected to emerge as the most lucrative over the forecast period.

Recent Developments

Some of the prominent players in the Gene therapy market include:

Segments Covered in the Report

This report forecasts revenue growth at global, regional, and country levels and provides an analysis of the latest industry trends in each of the sub-segments from 2021 to 2033. For this study, Nova one advisor, Inc. has segmented the global gene therapy market.

Indication

Vector Type

Route of Administration

By Region

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Gene Therapy Market Size Poised to Surge USD 52.40 Billion by 2033 - BioSpace

ZUG, Switzerland and BOSTON, April 22, 2024 (GLOBE NEWSWIRE) -- CRISPR Therapeutics(Nasdaq: CRSP), a biopharmaceutical company focused on creating transformative gene-based medicines for serious diseases, today announced an oral presentation highlighting the Company's lipid nanoparticle (LNP) approach for ocular editing will be presented at the American Society of Gene & Cell Therapy (ASGCT) 2024 Annual Meeting, taking place May 7 11, 2024, in Baltimore, MD and virtually.

The abstract describes our proprietary capabilities to deliver to and edit genes in the eye, opening a potential new focus area. Multiple LNPs as well as modified gRNAs and mRNAs were screened to achieve maximal editing in vivo. These optimized components have been applied to target myocilin (MYOC). Mutations of MYOC in trabecular meshwork cells have been linked to severe glaucomatous conditions. In human primary trabecular meshwork cells, up to 95% MYOC editing and 85% protein knockdown were seen. This novel approach aims to facilitate glaucoma treatment using transient expression of editing machinery targeting MYOC.

Title: Development of an In Vivo Non-Viral Ocular Editing Platform and Application to Potential Treatments for Glaucoma Session Type: In-Person Oral Presentation Session Title: Ophthalmic and Auditory: Delivery Innovations Abstract Number:87 Location: Room 318 323 Session Date and Time: Wednesday, May 8, 2024, 1:30 p.m. 3:15 p.m. ET

The accepted abstract is available online on the ASGCT website. The data are embargoed until 6:00 a.m. ET on the presentation day, Wednesday May 8, 2024. A copy of the presentation will be available at http://www.crisprtx.com once the presentation concludes.

About CRISPR Therapeutics Since its inception over a decade ago, CRISPR Therapeutics has transformed from a research-stage company advancing programs in the field of gene editing, to a company that recently celebrated the historic approval of the first-ever CRISPR-based therapy and has a diverse portfolio of product candidates across a broad range of disease areas including hemoglobinopathies, oncology, regenerative medicine, cardiovascular, autoimmune, and rare diseases. CRISPR Therapeutics advanced the first-ever CRISPR/Cas9 gene-edited therapy into the clinic in 2018 to investigate the treatment of sickle cell disease or transfusion-dependent beta thalassemia, and beginning in late 2023, CASGEVY (exagamglogene autotemcel) was approved in some countries to treat eligible patients with either of those conditions. The Nobel Prize-winning CRISPR science has revolutionized biomedical research and represents a powerful, clinically validated approach with the potential to create a new class of potentially transformative medicines. To accelerate and expand its efforts, CRISPR Therapeutics has established strategic partnerships with leading companies including Bayer and Vertex Pharmaceuticals. CRISPR Therapeutics AG is headquartered in Zug, Switzerland, with its wholly-owned U.S. subsidiary, CRISPR Therapeutics, Inc., and R&D operations based in Boston, Massachusetts and San Francisco, California, and business offices in London, United Kingdom. To learn more, visit http://www.crisprtx.com.

CRISPR Therapeutics Forward-Looking StatementThis press release may contain a number of forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995, as amended, including statements regarding CRISPR Therapeutics expectations about any or all of the following: (i) its ongoing and/or planned preclinical studies, clinical trials and pipeline products and programs, including, without limitation, the status of such studies and trials, potential expansion into new indications and expectations regarding data generally (including expected timing of data releases) as well as the data in the above-described abstract and any associated poster and the data that is being presented as described above; (ii) the safety, efficacy and clinical progress of its various clinical and preclinical programs including the program described in the oral presentation and poster; (iii) the data that will be generated by ongoing and planned preclinical studies and/or clinical trials, and the ability to use that data for the design and initiation of further preclinical studies and/or clinical trials; and (iv) the therapeutic value, development, and commercial potential of CRISPR/Cas9 gene editing technologies and therapies. Without limiting the foregoing, the words believes, anticipates, plans, expects and similar expressions are intended to identify forward-looking statements. You are cautioned that forward-looking statements are inherently uncertain. AlthoughCRISPR Therapeuticsbelieves that such statements are based on reasonable assumptions within the bounds of its knowledge of its business and operations, forward-looking statements are neither promises nor guarantees and they are necessarily subject to a high degree of uncertainty and risk. Actual performance and results may differ materially from those projected or suggested in the forward-looking statements due to various risks and uncertainties. These risks and uncertainties include, among others: the efficacy and safety results from ongoing pre-clinical studies and/or clinical trials will not continue or be repeated in ongoing or planned pre-clinical studies and/or clinical trials or may not support regulatory submissions;pre-clinical study and/or clinical trial results may not be favorable or support further development; one or more of its product candidate programs will not proceed as planned for technical, scientific or commercial reasons; future competitive or other market factors may adversely affect the commercial potential for its product candidates; uncertainties inherent in the initiation and completion of preclinical studies for its product candidates and whether results from such studies will be predictive of future results of future studies or clinical trials; uncertainties about regulatory approvals to conduct trials or to market products; uncertainties regarding the intellectual property protection for its technology and intellectual property belonging to third parties, and the outcome of proceedings (such as an interference, an opposition or a similar proceeding) involving all or any portion of such intellectual property; and those risks and uncertainties described under the heading "Risk Factors" in CRISPR Therapeutics most recent annual report on Form 10-K and in any other subsequent filings made byCRISPR Therapeuticswith theU.S. Securities and Exchange Commission, which are available on theSEC'swebsite atwww.sec.gov.CRISPR Therapeuticsdisclaims any obligation or undertaking to update or revise any forward-looking statements contained in this press release, other than to the extent required by law.

Investor Contact: Susan Kim +1-617-307-7503 susan.kim@crisprtx.com

Media Contact: Rachel Eides +1-617-315-4493 rachel.eides@crisprtx.com

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CRISPR Therapeutics to Present Oral Presentation at the American Society of Gene & Cell Therapy (ASGCT) 2024 ... - GlobeNewswire

CRISPR technologys initial applications were primarily focused on targeted genome editing, but its use has expanded into cell line engineering, which holds promises of groundbreaking advancements, insists Promega'sPhilip Hargreaves

CRISPR technology has come a long way since its discovery as a natural defence mechanism in bacteria. Scientists have harnessed its potential to precisely edit genes, paving the way for groundbreaking advancements in genetic engineering. Initially, CRISPR was primarily used for editing specific genes within organisms, but its scope has expanded rapidly to include the manipulation of entire cell lines. This ability to edit cell lines using CRISPR technology has unlocked a myriad of possibilities.

For many years researchers and drug developers have used immortalised cell lines (derived from humans but engineered to have stable properties over many divisions) to aid their work. These cell lines typically have the gene and/or protein target of interest expressed within them. CRISPR methodology has allowed quicker and more optimal manipulation of these cell lines.

As of now, CRISPR cell line technology is being used extensively for medical and agricultural applications. In medicine, researchers are employing CRISPR to engineer cell lines for therapeutic purposes, intending to treat diseases at the genetic level. For instance, CRISPR has been used to edit the genes of immune cells, enabling them to better target and eliminate cancer cells.

The Future Landscape

Looking ahead, the future of CRISPR cell line technology holds even more transformative possibilities. One of the key areas of exploration is regenerative medicine, where researchers aim to harness CRISPR to engineer cells for tissue repair and organ regeneration. The ability to precisely edit and manipulate cell lines could open new avenues for treating degenerative diseases and injuries, potentially revolutionising the field of transplantation.

The ability to precisely edit and manipulate cell lines could open new avenues for treating degenerative diseases and injuries, potentially revolutionising the field of transplantation

CRISPR technology is further poised to play a crucial role in the development of novel therapies for genetic disorders. The ability to edit problematic genes could pave the way for more effective treatments and even cures for conditions that were once considered incurable, such as Alzheimers.

Despite the remarkable progress in CRISPR technology, challenges and roadblocks still need to be addressed. Off-target effects, unintended mutations, and the potential for unpredictable consequences remain significant hurdles in developing and applying CRISPR-based therapies. Researchers are actively working to enhance the precision and safety of CRISPR technology to mitigate these risks.

CRISPR-engineered cell lines

One way to use CRISPR engineering to generate cell lines, which Promega is successfully offering, is to incorporate HiBiT technology. This 11 amino acid peptide can be fused to a target protein and serves as a luminescent tag. HiBiT can be integrated by knocking in the tag to the endogenous locus of the target using CRISPR gene editing, to help create a truer picture of protein behaviour and regulation in their natural cellular environment. HiBiT has a dynamic range spanning nine logs, which allows for the detection of extremely small quantities of tagged proteins.

In terms of applications, HiBiT's versatility is unmatched, as quantitative assays can be performed in both endpoint and live-cell formats, without the need for target-specific antibodies. From measuring target protein abundance to studying targeted protein degradation, protein secretion and receptor recycling, HiBiT opens up a myriad of possibilities in biomedical research. Its role in drug discovery is particularly noteworthy, enabling more precise and efficient screening of drug effects on cellular proteins.

The accessibility and affordability of CRISPR technology also need to be addressed to ensure that its benefits are not confined to a privileged few. Wide-scale adoption and integration of CRISPR cell line technology into various sectors will require collaborative efforts from researchers, policymakers, and the private sector. To aid in this endeavour, Promega now offers a comprehensive selection of pre-built CRISPR-edited cell line pools and clones, including HiBiT fusions.

Wide-scale adoption and integration of CRISPR cell line technology into various sectors will require collaborative efforts from researchers, policymakers, and the private sector

This development opens doors for researchers and developers as it reduces the costs involved in developing cell lines from scratch. Not only will this save budgets, but it also saves time. This means drug development times can be reduced by as much as 12 months, leading to vital medications being available much sooner.

The future of CRISPR cell line technology is undoubtedly exciting and promises transformative advancements in medicine, agriculture, and beyond. As scientists continue to unravel the mysteries of genetic engineering, there is a delicate balance between innovation and ethical stewardship to consider. But with efforts to maximise the technologys potential to make it more accessible and affordable, the future is bright.

Philip Hargreaves Ph.D, is director of strategic marketing & business development at Promega

Pic: Brano

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SNIPR Biome

SNIPR Biome receives funding from CARB-X to support advancement of CRISPR-medicine SNIPR001 into clinical trials in haematological cancer patients

Phase 1b/2a trial will evaluate SNIPR001 for the prevention of E.coli infections in patients undergoing hematopoietic stem cell transplantation

Copenhagen, April 22 2024: SNIPR Biome ApS (SNIPR), the company pioneering the development of precision medicines using CRISPR technology for microbial gene therapy, announces today that it has received $5.48 million from Combating Antibiotic-Resistant Bacteria Biopharmaceutical Accelerator (CARB-X) to co-fund a Phase 1b/2a clinical trial in hematological cancer patients.

The trial will evaluate SNIPR001, the first CRISPR-armed phage therapeutic that specifically targets E. coli in the gut, for the prevention of E. coli bloodstream infections in hematological cancer patients who are undergoing hematopoietic stem-cell transplantation (HSCT) and are colonized with Fluoroquinolone Resistant (FQR) E. coli. Fluoroquinolone is recommended in the US for prophylaxis of bacterial infections and febrile neutropenia in hematological cancer patients at high risk of neutropenia.

Despite the significant advances in hematologic cancer therapy over the past decade, infectious complications, and antimicrobial resistance (AMR) continue to pose significant threats to patients and clinical outcomes1. Currently, there are no approved therapies for the prevention of bloodstream infections (BSIs) in hematological cancer patients. SNIPR Biome is developing SNIPR001 to address this urgent unmet need to combat infections in hematological cancer patients.

Preclinical data published in Nature Biotechnology described SNIPR001s ability to selectively target and remove antibiotic-resistant E. coli strains in the gut, potentially offering a safe treatment which preserves the rest of the gut microbiome. This was supported by interim Phase 1 data published in 2023, which showed that oral dosing of SNIPR001 over seven days across three dosing levels in 24 healthy individuals was well tolerated. Furthermore, SNIPR001 could be recovered in faeces from treated individuals in a dose-dependent manner, and treatment with SNIPR001 numerically lowered gut E. coli levels.

Anticipated to begin later this year, the randomized, double-blinded Phase 1b/2a trial will investigate the safety, tolerability, pharmacokinetics, and pharmacodynamics of orally administrated SNIPR001 in 24 patients. It will be conducted at up to 10 sites across Europe and the United States.

CARB-X, a global non-profit partnership dedicated to supporting early-stage antibacterial research and development to address the rising threat of drug-resistant bacteria, has been a long-term collaborator with SNIPR in this field. The funding announced today enables SNIPR to move SNIPR001 into Phase 1b/2a clinical trials and will serve as a cornerstone for a further significant fundraise to enable the Company to continue development of its pipeline of CRISPR-based AMR and gut-directed gene therapies.

Story continues

Dr Christian Grndahl, Co-founder and CEO of SNIPR Biome, commented: Antibiotic resistance is one of healthcares biggest problems today, affecting treatment efficacy and survival among patients who are often already very sick. We are using our knowledge of gene editing and synthetic biology to create highly specific, designer bacteria and phage to disrupt, edit or add genes, and deliver these precision medicines in a carefully targeted way. We are pleased to be continuing our partnership with CARB-X who share our commitment to developing therapies for vulnerable patients.

Erin Duffy PhD, Chief of Research & Development, CARB-X, said: Having underscored safety for SNIPR001 in healthy subjects, SNIPR Biome is now focusing on demonstrating proof-of-mechanism for this novel product, with our support.We are keen to establish a link between gut decolonization and prevention of infection as a novel approach to antimicrobial resistance, and SNIPR001 offers the possibility of doing so.

CARB-X funding for this research is supported by the Biomedical Advanced Research and Development Authority under agreement number: 75A50122C00028, and by awards from Wellcome (WT224842), and Germanys Federal Ministry of Education and Research (BMBF). The content of this press release is solely the responsibility of the authors and does not necessarily represent the official views of CARB-X or any of its funders.

Ends

About SNIPR001 SNIPR001, a CRISPR-armed phage therapeutic that specifically targets E. coli in the gut, is designed to prevent infections from spreading into the bloodstream and represents a promising advancement against antibiotic-resistant pathogens. The pre-clinical studies of SNIPR001 published in Nature Biotechnology2 demonstrated the products activity against multi-drug resistant strains of E. coli and its specificity towards E. coli with no off-target effects toward any of the tested non-E. coli strains. SNIPR successfully completed a Phase 1 trial in the US, also funded by CARB-X, demonstrating safety of SNIPR001 and target engagement with E. coli in the gut of healthy subjects without disturbing the overall gut microbiome (NCT05277350), supporting its potential as a safe and effective preventative therapy for bloodstream infections in hematological cancer patients. SNIPR001 has been granted a Fast-Track designation for the indication Prophylaxis of bloodstream E. coli infections in patients with hematological malignancy at risk of neutropenia from the US Food and Drug Administration (FDA). SNIPR001 is also being developed to directly treat active E. coli infections.

About SNIPR BIOME SNIPR Biome is a Danish clinical-stage biotech company pioneering the development of precision medicines using CRISPR technology for microbial gene therapy. We are pioneering a novel use of CRISPR/Cas technology to better treat and prevent human diseases through precision killing of bacteria or gene modification. SNIPR Biome was the first company to orally dose humans with a CRISPR therapeutic and the first company to have been granted US and European patents for the use of CRISPR for targeting microbiomes. SNIPR technology is used in collaborations with Novo Nordisk A/S, CARB-X, SPRIN-D, and MD Anderson Cancer Center. For more information, visit http://www.sniprbiome.com and follow us on LinkedIn and X.

About CARB-X

CARB-X (Combating Antibiotic-Resistant Bacteria Biopharmaceutical Accelerator) is a global non-profit partnership dedicated to supporting early-stage antibacterial research and development to address the rising threat of drug-resistant bacteria. CARB-X supports innovative therapeutics, preventatives and rapid diagnostics. CARB-X is led by Boston University and funded by a consortium of governments and foundations. CARB-X funds only projects that target drug-resistant bacteria highlighted on the CDCs Antibiotic Resistant Threats list, or the Priority Bacterial Pathogens list published by the WHO, with a priority on those pathogens deemed Serious or Urgent on the CDC list or Critical or High on the WHO list. https://carb-x.org/ | X (formerly Twitter) @CARB_X

Contact ICR Consilium Tracy Cheung, Chris Welsh, Davide Salvi SNIPR@consilium-comms.com

SNIPR Biome Dr Christian Grndahl, Co-founder and CEO contact@sniprbiome.com http://www.sniprbiome.com

1 So M. Determining the Optimal Use of Antibiotics in Hematopoietic Stem Cell Transplant Recipients. JAMA Netw Open. 2023 Jun 1;6(6):e2317101 2 Gencay, Y.E., Jasinskyt, D., Robert, C. et al. Engineered phage with antibacterial CRISPRCas selectively reduce E. coli burden in mice. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-023-01759-y

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SNIPR Biome receives funding from CARB-X to support advancement of CRISPR-medicine SNIPR001 into clinical ... - Yahoo Finance

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Excerpt from:
Adeno-associated virus as a delivery vector for gene therapy of human diseases | Signal Transduction and Targeted ... - Nature.com

According to the latest research by nova one advisor, the global cell and gene therapy market size was valued at USD 18.13 billion in 2023 and is anticipated to reach around USD 97.33 billion by 2033, growing at a CAGR of 18.3% from 2024 to 2033.

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The cell and gene therapy market provides therapeutic solutions related to genes and cells. The market deals with research & development, testing, production, and distribution of products and treatment procedures related to genes and cells. Hospitals, research laboratories, pharmaceutical companies, pharmacies, research institutions, and universities are involved in delivering the applications associated with gene and cell therapies. Gene and cell therapies are developed to prevent, treat, or potentially cure numerous diseases. The potential of these therapies to cure, treat, or prevent diseases that are life-threatening increases the demand and boosts the growth of the market. Gene and cell therapies are used in blood stem cell transplantation, gene editing, engineering of the immune system, tissue regeneration, in-vivo gene transfer, cancer treatment, and treatment of different disorders. These therapies can provide better results and enhance quality of life.

North America dominated the cell and gene therapy market in 2023. North America is a developed region that has developed healthcare and research infrastructure, better facilities, and government support that boosts the growth of the market. Governments in the North American region have a huge national budget for healthcare and research. Countries like the U.S. and Canada contribute to the growth of the market in the North American region. As of now, the FDA has approved 37 products for gene and cell therapy. The U.S. has the American Society of Gene & Cell Therapy (ASGCT) for professionals, scientists, physicians, and patient advocates that help advance knowledge, education, and awareness for discovering and developing clinical applications of gene and cell therapy.

The Canadian government is also focusing on improving health with the help of genes and therapies and is launching various programs to help with this. The government launched Disruptive Technology Solutions, which will help tackle the challenges associated with gene and cell therapies. The treatment procedures will be done to cure rare genetic disorders and chronic diseases.

Key Takeaways:

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The cell and gene therapy market is exploding globally

Ground-breaking developments in next-generation cell and gene therapies (CGTs) offer curative value for patients with few to no other therapeutic interventions for either maintenance or cure within specific disease areas, many of which include rare and ultra-rare diseases.The largest therapeutic area is cancer, followed by musculoskeletal diseases and eye diseases.Multiple approved products have been launched in global markets and the number of clinical trials continues to grow. In Europe, these therapies are classified under Advanced Therapeutic Medicinal Products (ATMPs) and are driven by a diverse set of scientific advancements including CAR-T, TCR-T, stem cells, siRNA, oligonucleotides, gene editing (CRISPR, Zinc Fingers, TALENs) and viral transfection.

The global CGT market is projected to grow at a compound annual growth rate of over 36 percent from 2019-2025, to ~ 10 billion. With more than 900 companies globally focusing on CGTS and over 1,000 clinical trials being conducted, the industry could see numerous approvalsas many as 10 to 20 new advanced therapies per year starting in 2025. Moreover, 33% of these clinical trials is being conducted in Europe.1

Global biopharma companies as well as smaller, venture backed-up start-ups are rapidly investing in this complex space. In 2018, about $13 billion has been invested globally in advanced therapies such as cell, gene and gene modifying therapies. In 2019, 19 CGT-related M&A deals worth over $156 billion were completed.

As with any innovative and disruptive technology, CGT developers face challenges along several key stages of the product life cycle. Compared to chemical-based pharmaceuticals, key success factors such as enabling patient access, managing supply chain and manufacturing operations, evidencing compliance with increasingly complex regulatory requirements and alternate business models impose a greater burden.

Segments Insights:

By Therapy Insights

The market for cell and gene treatments consists of companies (organizations, sole proprietors, and partnerships) that sell the therapies they have developed. Cell therapy is the transfer of whole, living cells derived from allogeneic or autologous sources, while gene therapy is the introduction, deletion, or alteration of the genome to treat disease. The market is made up of the money that businesses creating goods for cell and gene therapy make from the sales of those items.

Cell treatment and gene therapy are the two primary product categories in this field. Gene therapy is a field of medicine that focuses on altering cells' genetic make-up to treat disease or reverse it by repairing or replacing genetic material that has been damaged. Oncology, dermatological, musculoskeletal, and other applications are among the many that are used in hospitals, ambulatory surgery centers, cancer treatment facilities, wound care facilities, and other industries.

Cell & Gene Therapy Market Revenue, By Therapy Type, 2022-2032 (USD Million)

By Therapy Type

2022

2023

2027

2031

2032

Cell Therapy

13,396.01

15,621.48

29,433.95

57,138.21

67,757.69

Gene Therapy

2,067.97

2,502.14

5,406.11

11,864.27

14,480.51

By Therapeutic class

Based on application, the market is divided into cardiovascular disease, cancer, genetic disorder, rare diseases, oncology, hematology, ophthalmology, infectious disease, neurological disorders. Among these, the infectious disease segment dominates the market in 2023. The oncological disorder segment held a revenue share of 13.53% in 2023. Research and treatment in the biomedical domains of cell therapy and gene therapy. Both treatments have the ability to lessen the underlying cause of hereditary disorders and acquired diseases. Both therapies aim to treat, prevent, or perhaps cure diseases. By repairing or changing specific cell types, or by employing cells to transport a medication across the body, cell therapy tries to treat diseases. Cell therapy involves growing or modifying cells outside of the body before injecting them into the patient. The cells may come from a donor (allogeneic cells) or the patient (autologous cells)6. By replacing, deactivating, or introducing genes into cells, either inside the body (in vivo) or outside the body, gene therapy seeks to treat disorders (ex vivo).

The market for genetic disorders is expanding as a result of factors like the high prevalence of genetic and chronic disease cases and the growing government initiatives to raise public knowledge of genetic testing and diagnosis. Researchers are developing novel techniques for screening, diagnosing, and treating patients for a variety of cardiac diseases as they investigate the genetic roots of heart and vascular illness. Some researchers are looking for new ways to nine patients who are at risk for sudden cardiac death. Others are examining how medicines that could postpone or obviate the need for cardiac surgery could benefit patients with uncommon illnesses.

The intricacy of mitochondrial genetics and the diverse clinical and biochemical symptoms of primary mitochondrial disorders (PMDs) have shown to be a significant obstacle to the development of effective disease-modifying medications. A successful clinical transition of genetic medicines for PMDs is possible, according to encouraging evidence from gene therapy trials in patients with Leber hereditary optic neuropathy and improvements in DNA editing tools.

Cell & Gene Therapy Market Revenue, By Therapeutic Class, 2022-2032 (USD Million)

By Therapeutic Class

2022

2023

2027

2031

2032

Cardiovascular Disease

744.36

882.84

1,780.08

3,697.84

4,460.03

Genetic Disorder

1,643.41

1,922.21

3,665.70

7,202.20

8,566.52

Oncology

1,936.87

2,272.26

4,385.58

8,720.66

10,403.81

Hematology

1,196.56

1,396.75

2,642.34

5,150.06

6,113.36

Ophthalmology

835.60

972.46

1,817.62

3,500.15

4,142.33

Infectious Disease

4,420.18

5,206.30

10,210.05

20,628.98

24,708.86

Neurological Disorders

658.61

777.29

1,536.51

3,129.23

3,755.58

Others

4,028.39

4,693.50

8,802.17

16,973.35

20,087.70

By Delivery Method

The market is split into In Vivo therapy and Ex Vivo therapy according to the type of therapy. In vivo therapy market is anticipated to grow exponentially throughout the projected period. When it comes to gene therapy, there are two different methods: ex vivo and in vivo, setting aside cell therapies. The altered human gene must first enter the diseased person's cells for gene therapy to take effect. There are two methods for doing this; Genetic material is supplied in vivo to afflicted cells (cancer cells or other cells) that are still present within an individual's body. After cells are collected and exposed to the genome in Ex vivo, altered genes are transferred to a person's body.

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Cell and Gene Therapy Market Size to Reach USD 97.33 Bn by 2033 - BioSpace

Hoxa9 and b-catenin molecules are a rare population of self-renewing HSCs found in bone marrow

Researchers from Kings College Londons (KCL) Comprehensive Cancer Centre have identified a key mechanism that governs how bone marrow stem cells work, which could potentially lead to new therapeutic pathways.

The findings from the study will help researchers further understand the key principles involved in stem cell biology and could provide new avenues for the development of efficient stem cell therapeutics.

Researchers identified two molecules, Hoxa9 and b-catenin, that control when bone marrow stem cells rest and recover, as well as when they act and replicate.

Both molecules work together to control a rare population of self-renewing stem cells that are predominantly found in bone marrow, known as haematopoietic stem cells (HSCs).

HSCs are protected from environmental stressors and prevent exhaustion by resting, while inactive HSCs must become active again, replicating themselves by turning into different blood cells, including red blood cells, white blood cells and platelets, to replenish the blood system and respond to problems including infections, blood loss and other complications.

Researchers discovered that this active/inactive characteristic of HSCs plays a key role in bone marrow transplantation, a vital procedure for several diseases, including cancer, as it is critical for cancer stem cells, which sustain the disease and cause relapse.

Researchers suggest that understanding this process will be vital when designing better treatments.

In addition, the team identified a critical enzyme known as PRMT1, which mediates the functions of the two molecules, offering a potential new avenue for the development of efficient stem cell therapeutics.

Eric So, professor and chair in Leukaemia Biology, Comprehensive Cancer Centre, School of Cancer and Pharmaceutical Sciences, KCL and study lead, commented: Given the critical functions of stem cells in bone marrow transplant and cancer biology, [the] identification of a new druggable pathway not only will help to better understand the stem cell biology but also facilitates the development of more effective therapeutics in the future.

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Key mechanism controlling bone marrow stem cells could lead to new therapies - PharmaTimes - PharmaTimes

ZUG, Switzerland and BOSTON, April 01, 2024 (GLOBE NEWSWIRE) -- CRISPR Therapeutics(Nasdaq: CRSP), a biopharmaceutical company focused on creating transformative gene-based medicines for serious diseases, today announced an oral presentation at the American Society of Gene & Cell Therapy (ASGCT) 2024 Annual Meeting, taking place May 7 11, 2024, in Baltimore, MD and virtually.

Title: Development of an In Vivo Non-Viral Ocular Editing Platform and Application to Potential Treatments for Glaucoma Session Type: In-Person Oral Presentation Session Title: Ophthalmic and Auditory: Delivery Innovations Abstract Number:87 Location: Room 318 323 Session Date and Time: Wednesday, May 8, 2024, 1:30 p.m. 3:15 p.m. ET

Abstracts will be released to the public on April 22, 2024, at 4:30 p.m. ET at https://annualmeeting.asgct.org/. The data are embargoed until 6:00 a.m. ET on the presentation day, Wednesday May 8, 2024. A copy of the presentation will be available at http://www.crisprtx.com once the presentation concludes.

About CRISPR Therapeutics Since its inception over a decade ago, CRISPR Therapeutics has transformed from a research-stage company advancing programs in the field of gene editing, to a company with a diverse portfolio of product candidates across a broad range of disease areas including hemoglobinopathies, oncology, regenerative medicine, cardiovascular and rare diseases. The Nobel Prize-winning CRISPR science has revolutionized biomedical research and represents a powerful, clinically validated approach with the potential to create a new class of potentially transformative medicines. To accelerate and expand its efforts, CRISPR Therapeutics has established strategic partnerships with leading companies including Bayer and Vertex Pharmaceuticals. CRISPR Therapeutics AG is headquartered in Zug, Switzerland, with its wholly-owned U.S. subsidiary, CRISPR Therapeutics, Inc., and R&D operations based in Boston, Massachusetts and San Francisco, California, and business offices in London, United Kingdom. To learn more, visit http://www.crisprtx.com.

Investor Contact: Susan Kim +1-617-307-7503 susan.kim@crisprtx.com

Media Contact: Rachel Eides +1-617-315-4493 rachel.eides@crisprtx.com

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CRISPR Therapeutics to Present at the American Society of Gene & Cell Therapy (ASGCT) 2024 Annual Meeting - GlobeNewswire

The global genetic analysis market was evaluated at USD 10.55 billion in 2023 and is expected to attain around USD 23.60 billion by 2033, growing at a CAGR of 8.39% from 2024 to 2033. The increasing demand for genetic testing services is driving growth within the genetic analysis market.

Market Overview

The genetic analysis market is experiencing significant transformation due to advances in genetic technology, which are fundamentally changing perceptions and practices within the healthcare industry. At the heart of this transformation lies the process of genetic analysis, which involves the examination of DNA samples to identify mutations that may influence disease susceptibility or treatment response. This analysis is pivotal for understanding the structure and function of genes, with techniques such as gene cloning playing a crucial role in isolating and replicating specific genes for detailed examination.

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One notable aspect of genetic analysis is its diverse clinical applications. It serves as a diagnostic tool, aiding in the confirmation of diagnoses in symptomatic individuals, while also facilitating the monitoring of disease prognosis and treatment response. Additionally, genetic analysis enables predictive or predisposition testing, allowing for the identification of individuals at risk of developing certain diseases before symptoms manifest.

The emergence of predictive genetic testing is creating new market opportunities, as it enables proactive disease prevention strategies and early interventions. As perceptions regarding genetic testing continue to evolve, the market for genetic analysis is expected to witness sustained growth, driven by its potential to revolutionize patient care and improve health outcomes.

Key Insights

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North America to sustain its position in the upcoming years with the U.S. being largest contributor

In 2023, North America emerged as the dominant force in the genetic analysis market, particularly in the United States. The US showcased a robust infrastructure with 200 laboratories actively conducting 37,124 clinical tests, underscoring the region's significant investment and adoption of genetic analysis technologies. Notably, 29 laboratories specialized in whole exome sequencing (WES), while 17 laboratories focused on whole genome sequencing (WGS), indicating a wide array of genetic testing capabilities available within the country.

The United States exhibits a proactive approach towards healthcare, as evidenced by mandatory newborn screening programs targeting a specific set of genetic diseases. Although the exact set of diseases screened may vary from state to state, the emphasis remains on conditions where early diagnosis is crucial for effective treatment or prevention strategies. This regulatory framework underscores the importance placed on leveraging genetic analysis for proactive healthcare management and disease prevention initiatives.

Beyond clinical applications, genetic analysis in North America extends to ecological and environmental contexts. The presence of invasive species such as Phragmites australis subsp. australis poses ecological challenges across multiple regions. The co-occurrence of this invasive subspecies with native counterparts and instances of hybridization necessitates precise differentiation methods for effective management strategies. Genetic analysis plays a pivotal role in distinguishing between phragmites subspecies or haplotypes, facilitating targeted management efforts to mitigate ecological harm and preserve native ecosystems.

Asia Pacific to witness lucrative opportunities in the upcoming years

Asia Pacific emerges as a pivotal region poised for substantial growth in the genetic analysis sector, driven by dynamic developments in genetic counselling and genome mapping initiatives. Forecasts indicate that Asia Pacific will experience the fastest growth rate in the genetic analysis market during the forecast period, underscoring the region's significance in shaping the future of genetic healthcare services.

A recent milestone in the region's genetic counselling landscape is the establishment of the Professional Society of Genetic Counsellors in Asia (PSGCA). Formed as a special interest group of the Asia Pacific Society of Human Genetics, PSGCA aims to spearhead the advancement and integration of the genetic counselling profession across Asia. With a vision to become the premier organization driving genetic counselling mainstream adoption in the region, PSGCA endeavors to ensure equitable access to genetic counselling services for individuals. Its mission centers on elevating standards of practice, curriculum, research, and continuing education to promote quality genetic counselling services throughout Asia.

The rapid evolution of genetic and genomic technologies has significantly transformed healthcare services in low- and middle-income countries (LMICs) across the Asia-Pacific region. Initially focused on population-based disease prevention strategies, genetic services have transitioned towards clinic-based and therapeutics-oriented approaches. Notably, the region's genetic diversity, exemplified by populous and genetically varied countries such as China, India, Japan, and Indonesia, positions them as prime candidates for genome mapping research endeavors.

How the genetic analysis market in Asia Pacific

Report Highlights

By Product

The reagents & kits segment asserted dominance in the genetic analysis market in 2023. DNA reagents play a pivotal role in various DNA-related processes and techniques, including sequencing, synthesis, cloning, and mutagenesis. These products encompass a diverse range, such as plasmids, buffers, labeling technology, columns, and comprehensive test kits utilized in DNA testing, including direct-to-consumer (DTC) genetic tests. While offering accessible information about the scientific basis of tests, the usage of DTC genetic tests carries inherent risks due to the absence of personalized guidance concerning the results.

The instruments segment emerged as the fastest-growing sector within the genetic analysis market. Core laboratory instruments constitute essential tools in genetic engineering research, facilitating precise and reliable experimentation. Polymerase Chain Reaction (PCR) machines, also known as thermal cyclers, stand as indispensable equipment in genetic engineering labs, enabling the amplification of specific DNA segments crucial for detailed analysis.

By Test

In 2023, the disease diagnostic testing segment emerged as the dominant force in the genetic analysis market. This segment specializes in identifying whether individuals harbor specific genetic diseases by detecting alterations in particular genes. While these tests excel at pinpointing gene mutations, they often fall short in determining disease severity or age of onset. Thousands of diseases stem from mutations in a single gene, making diagnostic testing pivotal in confirming or ruling out genetic diseases and chromosomal abnormalities. Frequently utilized during pregnancy or when symptomatic, diagnostic genetic testing offers crucial insights for accurate diagnosis and timely intervention.

The prenatal and newborn testing segment emerged as the fastest-growing sector in the genetic analysis market during the forecast period. Prenatal genetic testing provides prospective parents with vital information regarding potential genetic disorders in the fetus. Prenatal screening tests assess the likelihood of fetal aneuploidy and select disorders, while prenatal diagnostic tests definitively ascertain the presence of specific disorders. These tests, conducted on fetal or placental cells obtained through procedures like amniocentesis or chorionic villus sampling (CVS), play a pivotal role in informed decision-making during pregnancy.

Newborn screening, a subset of prenatal and newborn testing, comprises a set of laboratory tests performed on newborns to detect known genetic diseases. Typically conducted via a heel prick within the first few days of life, newborn screening enables early identification and intervention for treatable genetic conditions, thereby improving health outcomes. As the demand for early detection and preventive measures rises, the prenatal and newborn testing segment is poised for continued growth, bolstering the comprehensive landscape of genetic analysis.

By Technology

In 2023, the real-time PCR system segment emerged as the dominant force in the genetic analysis market. Real-time PCR (RT-PCR) systems offer unparalleled capabilities for quantitative genotyping and detection of single nucleotide polymorphisms (SNPs), allelic discrimination, and genetic variations even in samples with minimal mutation carriers. Multiplex PCR systems, a subset of RT-PCR, are gaining prominence, particularly in plant/microbe associations, where standard PCR methods prove inadequate. Multiplex RT-PCR facilitates the identification of multiple genes through the utilization of fluorochromes and analysis of melting curves, providing enhanced accuracy and efficiency in genetic analysis.

The next-generation sequencing (NGS) segment emerged as the fastest-growing sector in the genetic analysis market. NGS technology revolutionizes DNA sequencing and RNA sequencing and variant/mutation detection by enabling high-throughput sequencing of hundreds to thousands of genes or whole genomes within a short timeframe. The sequence variants/mutations detected by NGS hold profound implications for disease diagnosis, prognosis, therapeutic decision-making, and patient follow-up, paving the way for personalized precision medicine initiatives.

By Application

In 2023, the infectious diseases segment asserted dominance in the genetic analysis market, offering molecular genetic tests capable of identifying common viruses or bacteria responsible for respiratory infections and infectious diarrhea. These tests, conducted on samples collected from the nose and throat or a single stool sample, facilitate rapid and accurate diagnosis, enabling timely treatment and containment of infectious outbreaks.

The genetic diseases segment emerged as the fastest-growing sector in the genetic analysis market during the forecast period. The extent to which genes contribute to diseases varies, presenting opportunities for advancements in understanding genetic mechanisms underlying various conditions. This progress facilitates the development of early diagnostic tests, novel treatments, and preventive interventions to mitigate disease onset or severity.

By End Use

In 2023, the research & development laboratories segment emerged as the dominant force in the genetic analysis market, actively driving advancements in genetic disease study and testing technology. These laboratories are pivotal in enhancing clinical patient care by conducting rigorous research and development activities aimed at improving test strategies and introducing novel genetic tests. Board-certified directors and genetic counsellors collaborate closely with laboratory supervisors and technologists to ensure the delivery of accurate and reliable results within stipulated timelines. With a focus on meeting stringent validation standards, approved tests undergo thorough evaluations of methodology and clinical utility. Research programs within these laboratories leverage collective expertise to propel the field of genetics and genetic testing forward.

The diagnostic centers segment is poised for significant growth in the genetic analysis market during the forecast period. Diagnostic centers offer a comprehensive range of testing services crucial for diagnosing diverse medical conditions. By providing accurate and informed diagnoses, diagnostic centers enable physicians to develop effective treatment plans, ultimately enhancing patient outcomes. Leveraging advanced diagnostic technologies and techniques, these centers play a vital role in identifying underlying causes of diseases, monitoring disease progression, and devising personalized treatment approaches. Collaborating with healthcare providers like primary care physicians, specialists, and hospitals, diagnostic centers ensure accurate and timely diagnoses across a spectrum of medical conditions, reinforcing their indispensable role in modern healthcare delivery.

Market Dynamics

Driver: Advances in Genetic Sequencing and Gene Therapy

Significant strides in genetic sequencing, human genome analysis, and medical genetics have revolutionized disease understanding, diagnostic accuracy, and drug development targets. A pivotal breakthrough in medical genetics is the emergence of gene therapy, which involves modifying or replacing genes to treat or prevent diseases. Already applied successfully in treating conditions like inherited blindness and severe combined immunodeficiency (SCID), gene therapy is poised to expand its impact further.

Future projections indicate that gene therapy will play an increasingly vital role in medical genetics, offering treatments for previously untreatable diseases. This trajectory is expected to fuel the growth of the genetic analysis market, as the demand for advanced genetic testing and analysis escalates to support the development and implementation of gene therapy treatments.

Restraint: Privacy Concerns in Genetic Analysis

Privacy concerns poses a major challenge in the genetic analysis domain due to the inherent uniqueness of genomic data, hindering true anonymization efforts. Additionally, security measures are crucial to restrict access to data based on authorized clearance levels, safeguarding against unauthorized breaches. Confidentiality emerges as a key ethical consideration, dictating the responsible sharing of genetic data. These privacy concerns, among others, including consent and data ownership, serve as significant restraints in the genetic analysis market. Addressing these challenges effectively is essential to ensure ethical practices and foster trust among stakeholders, thereby mitigating the barriers to market growth.

Opportunity: Integration of Artificial Intelligence in Genetic Analysis

The integration of artificial intelligence (AI) is revolutionizing clinical genetics, offering unprecedented opportunities for advancement. AI algorithms possess the capability to analyse vast volumes of genetic data rapidly and accurately, facilitating more precise diagnoses and tailored treatment plans. Furthermore, AI empowers predictive analysis of disease risk, enabling the development of proactive disease prevention strategies. In genetic engineering and gene therapy research, AI serves as a powerful tool, aiding in hypothesis generation and experimental techniques. Leveraging AI, researchers can detect hereditary and gene-related disorders with greater efficiency.

Moreover, AI-driven developments hold immense promise for rational drug discovery and design, ultimately impacting humanity's well-being. As AI and machine learning (ML) technologies continue to drive innovation in drug development, genetics emerges as a prime beneficiary, with AI expected to influence every facet of the human experience. This presents a compelling opportunity for the genetic analysis market to capitalize on AI-driven advancements and propel transformative growth.

Recent Developments

Key Players in the Clinical Trials Market

Segments Covered in the Report

By Product

By Test

By Technology

By Application

By End-use

By Geography

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Genetic Analysis Market Size to Attain Around USD 23.60 BN by 2033 - BioSpace

Summary: Alzheimers disease, traditionally seen as a brain-centric condition, may have systemic origins and can be accelerated through bone marrow transplants from donors with familial Alzheimers to healthy mice.

A new study underscores the diseases potential transmission via cellular therapies and suggests screening donors for Alzheimers markers to prevent inadvertent disease transfer.

By demonstrating that amyloid proteins from peripheral sources can induce Alzheimers in the central nervous system, this research shifts the understanding of Alzheimers towards a more systemic perspective, highlighting the need for cautious screening in transplants and blood transfusions.

Key Facts:

Source: Cell Press

Familial Alzheimers disease can be transferred via bone marrow transplant, researchers show March 28 in the journalStem Cell Reports. When the team transplanted bone marrow stem cells from mice carrying a hereditary version of Alzheimers disease into normal lab mice, the recipients developed Alzheimers diseaseand at an accelerated rate.

The study highlights the role of amyloid that originates outside of the brain in the development of Alzheimers disease, which changes the paradigm of Alzheimers from being a disease that is exclusively produced in the brain to a more systemic disease.

Based on their findings, the researchers say that donors of blood, tissue, organ, and stem cells should be screened for Alzheimers disease to prevent its inadvertent transfer during blood product transfusions and cellular therapies.

This supports the idea that Alzheimers is a systemic disease where amyloids that are expressed outside of the brain contribute to central nervous system pathology, says senior author and immunologist Wilfred Jefferies, of the University of British Columbia.

As we continue to explore this mechanism, Alzheimers disease may be the tip of the iceberg and we need to have far better controls and screening of the donors used in blood, organ and tissue transplants as well as in the transfers of human derived stem cells or blood products.

To test whether a peripheral source of amyloid could contribute to the development of Alzheimers in the brain, the researchers transplanted bone marrow containing stem cells from mice carrying a familial version of the diseasea variant of the human amyloid precursor protein (APP) gene, which, when cleaved, misfolded and aggregated, forms the amyloid plaques that are a hallmark of Alzheimers disease.

They performed transplants into two different strains of recipient mice: APP-knockout mice that lacked an APP gene altogether, and mice that carried a normal APP gene.

In this model of heritable Alzheimers disease, mice usually begin developing plaques at 9 to 10 months of age, and behavioral signs of cognitive decline begin to appear at 11 to 12 months of age. Surprisingly, the transplant recipients began showing symptoms of cognitive decline much earlierat 6 months post-transplant for the APP-knockout mice and at 9 months for the normal mice.

The fact that we could see significant behavioral differences and cognitive decline in the APP-knockouts at 6 months was surprising but also intriguing because it just showed the appearance of the disease that was being accelerated after being transferred, says first author Chaahat Singh of the University of British Columbia.

In mice, signs of cognitive decline present as an absence of normal fear and a loss of short and long-term memory. Both groups of recipient mice also showed clear molecular and cellular hallmarks of Alzheimers disease, including leaky blood-brain barriers and buildup of amyloid in the brain.

Observing the transfer of disease in APP-knockout mice that lacked an APP gene altogether, the team concluded that the mutated gene in the donor cells can cause the disease and observing that recipient animals that carried a normal APP gene are susceptible to the disease suggests that the disease can be transferred to health individuals.

Because the transplanted stem cells were hematopoietic cells, meaning that they could develop into blood and immune cells but not neurons, the researchers demonstration of amyloid in the brains of APP knockout mice shows definitively that Alzheimers disease can result from amyloid that is produced outside of the central nervous system.

Finally the source of the disease in mice is a human APP gene demonstrating the mutated human gene can transfer the disease in a different species.

In future studies, the researchers plan to test whether transplanting tissues from normal mice to mice with familial Alzheimers could mitigate the disease and to test whether the disease is also transferable via other types of transplants or transfusions and to expand the investigation of the transfer of disease between species.

In this study, we examined bone marrow and stem cells transplantation. However, next it will be important to examine if inadvertent transmission of disease takes place during the application of other forms of cellular therapies, as well as to directly examine the transfer of disease from contaminated sources, independent from cellular mechanisms, says Jefferies.

Funding:

This research was supported by the Canadian Institutes of Health Research, the W. Garfield Weston Foundation/Weston Brain Institute, the Centre for Blood Research, the University of British Columbia, the Austrian Academy of Science, and the Sullivan Urology Foundation at Vancouver General Hospital.

Author: Kristopher Benke Source: Cell Reports Contact: Kristopher Benke Cell Reports Image: The image is credited to Neuroscience News

Original Research: The findings will appear in Stem Cell Reports

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Hereditary Alzheimer's Transmitted Via Bone Marrow Transplants - Neuroscience News

Figure 1: Total deal value by deal therapy area of licensing agreements for innovator drugs utilising the CRISPR system, globally, 2019-2024, year to date. Credit: GlobalData.

Licensing agreements for innovator drugs utilising clusteredregularly interspaced short palindromic repeats (CRISPR) technologies saw oncology, immunology, and central nervous system as the top three therapy areas by total deal value with a combined $21bn over the past five years.

Furthermore, haematological disorders saw almost three times more licensing agreement deal value in 2022 compared to 2020, reaching a total deal value of $1.8bn in the past five years (Figure 1), as reported by GlobalDatas Pharma Intelligence Center Deals Database.

This highlights the growing importance of advancements in CRISPR for haematological disorder therapies.

In December 2023, the US Food and Drug Administrations approval of Casgevy, the first CRISPR and CRISPR-associated protein 9 (Cas9) genome editing therapy developed by Vertex Pharmaceuticals and CRISPR Therapeutics for sickle cell disease and beta thalassemia represented a major milestone in gene therapy.

Casgevy precisely edits DNA in blood stem cells by utilising CRISPR/Cas9 technology, involving taking the patients bone marrow stem cells and enhancing their expression of fetal haemoglobin before reintroducing these edited stem cells back into the patient.

This restores healthy haemoglobin production in patients with sick cell disease and beta thalassemia, effectively alleviating the symptoms of these diseases.

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Figure 1 shows innovator drugs harnessing CRISPR systems saw 182% growth in total licensing agreement deal value from $5.6bn in 2020 to $15.8bn in 2022, according to GlobalDatas Pharma Intelligence Center Deals Database.

Oncology represented more than half of the total deal value for the top three therapy areas with $11.9bn, followed by immunology with $6.7bn, and central nervous system with $2.3bn, according to GlobalDatas Pharma Intelligence Center Deals Database.

Pharma giants such as Lily and Sanofi have recently partnered with companies developing CRISPR-based technologies.

Last year, Prevail Therapeutics, a subsidiary of Lily, secured exclusive rights to Scribe Therapeutics CRISPR X-Editing (XE) technologies for $1.65bn.

This licensing agreement, aimed at developing genetic therapies for neurological and neuromuscular diseases, stands as the largest CRISPR-based deal of the year.

Concurrently, Sanofi expanded its collaboration with Scribe in July 2023, with a deal worth up to $1.24bn, focusing on leveraging Scribes XE genome editing technologies for the development of in vivo therapies, particularly sickle cell disease and other genomic disorders.

Moreover, Lilys expertise in cardiometabolic diseases prompted the company to partner with Beam Therapeutics in October last year.

This agreement, valued at up to $600m, involved acquiring rights held by Beam in Verve Therapeutics, a gene-editing company focused on single-course gene editing therapies for cardiovascular disease.

This includes Verves programmes targeting PCSK9 and ANGPTL3, both set for clinical initiation this year.

CRISPR technology is revolutionising targeted gene therapies for various unmet diseases by precisely targeting diverse genomic sites.

This advancement in precision medicine offers hope for more tailored treatments and improved patient outcomes.

Furthermore, the growing number of CRISPR-based therapies in clinical trials is expected to fuel significant interest and drive further progress in this field.

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CRISPR drug licensing deals secure $21bn in top three therapy areas over five years - Pharmaceutical Technology

Abstract

Genome editing is the modification of genomic DNA at a specific target site in a wide variety of cell types and organisms, including insertion, deletion and replacement of DNA, resulting in inactivation of target genes, acquisition of novel genetic traits and correction of pathogenic gene mutations. Due to the advantages of simple design, low cost, high efficiency, good repeatability and short-cycle, CRISPR-Cas systems have become the most widely used genome editing technology in molecular biology laboratories all around the world. In this review, an overview of the CRISPR-Cas systems will be introduced, including the innovations, the applications in human disease research and gene therapy, as well as the challenges and opportunities that will be faced in the practical application of CRISPR-Cas systems.

Keywords: CRISPR, Cas9, Genome editing, Human disease models, Rabbit, Gene therapy, Off target effects

Genome editing is the modification of genomic DNA at a specific target site in a wide variety of cell types and organisms, including insertion, deletion and replacement of DNA, resulting in inactivation of target genes, acquisition of novel genetic traits and correction of pathogenic gene mutations [1], [2], [3]. In recent years, with the rapid development of life sciences, genome editing technology has become the most efficient method to study gene function, explore the pathogenesis of hereditary diseases, develop novel targets for gene therapy, breed crop varieties, and so on [4], [5], [6], [7].

At present, there are three mainstream genome editing tools in the world, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the RNA-guided CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) nucleases systems [8], [9], [10]. Due to the advantages of simple design, low cost, high efficiency, good repeatability and short-cycle, CRISPR-Cas systems have become the most widely used genome editing technology in molecular biology laboratories all around the world [11], [12]. In this review, an overview of the CRISPR-Cas systems will be introduced, including the innovations and applications in human disease research and gene therapy, as well as the challenges and opportunities that will be faced in the practical application of CRISPR-Cas systems.

CRISPR-Cas is an adaptive immune system existing in most bacteria and archaea, preventing them from being infected by phages, viruses and other foreign genetic elements [13], [14]. It is composed of CRISPR repeat-spacer arrays, which can be further transcribed into CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), and a set of CRISPR-associated (cas) genes which encode Cas proteins with endonuclease activity [15]. When the prokaryotes are invaded by foreign genetic elements, the foreign DNA can be cut into short fragments by Cas proteins, then the DNA fragments will be integrated into the CRISPR array as new spacers [16]. Once the same invader invades again, crRNA will quickly recognize and pair with the foreign DNA, which guides Cas protein to cleave target sequences of foreign DNA, thereby protecting the host [16].

CRISPR-Cas systems can be classified into 2 classes (Class 1 and Class 2), 6 types (I to VI) and several subtypes, with multi-Cas protein effector complexes in Class 1 systems (Type I, III, and IV) and a single effector protein in Class 2 systems (Type II, V, and VI) [17], [18]. The classification, representative members, and typical characteristics of each CRISPR-Cas system are summarized in [10], [12], [15], [16], [17], [18].

Summary of CRISPR-Cas systems.

Type II CRISPR-Cas9 system derived from Streptococcus pyogenes (SpCas9) is one of the best characterized and most commonly used category in numerous CRISPR-Cas systems [18], [19]. The main components of CRISPR-Cas9 system are RNA-guided Cas9 endonuclease and a single-guide RNA (sgRNA) [20]. The Cas9 protein possesses two nuclease domains, named HNH and RuvC, and each cleaves one strand of the target double-stranded DNA [21]. A single-guide RNA (sgRNA) is a simplified combination of crRNA and tracrRNA [22]. The Cas9 nuclease and sgRNA form a Cas9 ribonucleoprotein (RNP), which can bind and cleave the specific DNA target [23]. Furthermore, a protospacer adjacent motif (PAM) sequence is required for Cas9 proteins binding to the target DNA [20].

During genome editing process, sgRNA recruits Cas9 endonuclease to a specific site in the genome to generate a double-stranded break (DSB), which can be repaired by two endogenous self-repair mechanisms, the error-prone non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway [24]. Under most conditions, NHEJ is more efficient than HDR, for it is active in about 90% of the cell cycle and not dependent on nearby homology donor [25]. NHEJ can introduce random insertions or deletions (indels) into the cleavage sites, leading to the generation of frameshift mutations or premature stop codons within the open reading frame (ORF) of the target genes, finally inactivating the target genes [26], [27]. Alternatively, HDR can introduce precise genomic modifications at the target site by using a homologous DNA repair template [28], [29] (). Furthermore, large fragment deletions and simultaneous knockout of multiple genes could be achieved by using multiple sgRNAs targeting one single gene or more [30], [31].

Mechanism of genome editing. Double-strand break (DSB) induced by nucleases can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. NHEJ can introduce random insertions or deletions (indels) of varying length at the site of the DSB. Alternatively, HDR can introduce precise genomic modifications at the target site by using a homologous DNA donor template.

CRISPR-Cas systems have become the most favorite genome editing tool in the molecular biology laboratory since they were confirmed to have genome editing capabilities in 2012 [23]. They have made numerous achievements in the field of correcting pathogenic mutations, searching for essential genes for cancer immunotherapy, and solving key problems in organ xenotransplantation [5], [32], [33]. Unfortunately, there are still some limitations which need to solve in CRISPR-Cas systems, such as potential off-target effects, limited genome-targeting scope restricted by PAM sequences, and low efficiency and specificity [34], [35]. Therefore, many research teams have been trying to improve this tool.

By introducing two point mutations, H840A and D10A, into HNH and RuvC nuclease domain, researchers have obtained a nuclease dead Cas9 (dCas9) [36]. The dCas9 lacks DNA cleavage activity, but DNA binding activity is not affected. Then, by fusing transcriptional activators or repressors to dCas9, the CRISPR-dCas9 system can be used to activate (CRISPRa) or inhibit (CRISPRi) transcription of target genes [37], [38]. Additionally, dCas9 can be fused to various effector domains, which enables sequence-specific recruitment of fluorescent proteins for genome imaging and epigenetic modifiers for epigenetic modification [39], [40]. Furthermore, this system is easy to operate and allows simultaneous manipulation of multiple genes within a cell [38].

In order to improve the efficiency of site-directed mutagenesis, base editing systems containing dCas9 coupled with cytosine deaminase (cytidine base editor, CBE) or adenosine deaminase (adenine base editor, ABE) have been developed [41], [42]. It can introduce CG to TA or AT to GC point mutations into the editing window of the sgRNA target sites without double-stranded DNA cleavage [41], [42]. Since base editing systems avoid the generation of random insertions or deletions to a great extent, the results of gene mutation are more predictive. However, owing to the restriction of base editing window, base editing systems are not suitable for any target sequence in the genome. Accordingly, C-rich sequences, for example, would produce a lot of off-target mutations [43]. Therefore, researchers have always been trying to develop and optimize novel base editing systems to overcome this drawback [44]. At present, base editing systems have been widely used in various cell lines, human embryos, bacteria, plants and animals for efficient site-directed mutagenesis, which may have broad application prospects in basic research, biotechnology and gene therapy [45], [46], [47]. In theory, 3956 gene variants existing in Clin var database could be repaired by base substitution of C-T or G-A [42], [48].

An NGG PAM at the 3 end of the target DNA site is essential for the recognization and cleavage of the target gene by Cas9 protein [20]. Besides classical NGG PAM sites, other PAM sites such as NGA and NAG also exist, but their efficiency of genome editing is not high [49]. However, such PAM sites only exist in about one-sixteenth of the human genome, thereby largely restricting the targetable genomic loci. For this purpose, several Cas9 variants have been developed to expand PAM compatibility.

In 2018, David Liu et al.[50] developed xCas9 by phage-assisted continuous evolution (PACE), which can recognize multiple PAMs (NG, GAA, GAT, etc.). In the latter half of the same year, Nishimasu et al. developed SpCas9-NG, which can recognize relaxed NG PAMs [51]. In 2020, Miller et al. developed three new SpCas9 variants recognizing non-G PAMs, such as NRRH, NRCH and NRTH PAMs [52]. Later in the same year, Walton et al. developed a SpCas9 variant named SpG, which is capable of targeting an expanded set of NGN PAMs [53]. Subsequently, they optimized the SpG system and developed a near-PAMless variant named SpRY, which is capable of editing nearly all PAMs (NRN and NYN PAMs) [53].

By using these Cas9 variants, researchers have repaired some previously inaccessible disease-relevant genetic variants [51], [52], [53]. However, there are still some drawbacks in these variants, such as low efficiency and cleavage activity [50], [51]. Therefore, they should be further improved by molecular engineering in order to expand the applications of SpCas9 in disease-relevant genome editing.

In addition to editing DNA, CRISPR-Cas systems can also edit RNA. Class 2 Type VI CRISPR-Cas13 systems contain a single RNA-guided Cas13 protein with ribonuclease activity, which can bind to target single-stranded RNA (ssRNA) and specifically cleave the target [54]. To date, four Cas13 proteins have been identified: Cas13a (also known as C2c2), Cas13b, Cas13c and Cas13d [55]. They have successfully been applied in RNA knockdown, transcript labeling, splicing regulation and virus detection [56], [57], [58]. Later, Feng Zhang et al. developed two RNA base edting systems (REPAIR system, enables A-to-I (G) replacement; RESCUE system, enables C-to-U replacement) by fusing catalytically inactivated Cas13 (dCas13) with the adenine/cytidine deaminase domain of ADAR2 (adenosine deaminase acting on RNA type 2) [59], [60].

Compared with DNA editing, RNA editing has the advantages of high efficiency and high specificity. Furthermore, it can make temporary, reversible genetic edits to the genome, avoiding the potential risks and ethical issues caused by permanent genome editing [61], [62]. At present, RNA editing has been widely used for pre-clinical studies of various diseases, which opens a new era for RNA level research, diagnosis and treatment.

Recently, Anzalone et al. developed a novel genome editing technology, named prime editing, which can mediate targeted insertions, deletions and all 12 types of base substitutions without double-strand breaks or donor DNA templates [63]. This system contains a catalytically impaired Cas9 fused to a reverse transcriptase and a prime editing guide RNA (pegRNA) with functions of specifying the target site and encoding the desired edit [63]. After Cas9 cleaves the target site, the reverse transcriptase uses pegRNA as a template for reverse transcription, and then, new genetic information can be written into the target site [63]. Prime editing can effectively improve the efficiency and accuracy of genome editing, and significantly expand the scope of genome editing in biological and therapeutic research. In theory, it is possible to correct up to 89% known disease-causing gene mutations [63]. Nevertheless, as a novel genome editing technique, more research is still needed to further understand and improve prime editing system.

So far, as a rapid and efficient genome editing tool, CRISPR-Cas systems have been extensively used in a variety of species, including bacteria, yeast, tobacco, Arabidopsis, sorghum, rice, Caenorhabditis elegans, Drosophila, zebrafish, Xenopus laevis, mouse, rat, rabbit, dog, sheep, pig and monkey [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], as well as various human cell lines, such as tumor cells, adult cells and stem cells [79], [80]. In medical field, the most important application of CRISPR-Cas systems is to establish genetically modified animal and cell models of many human diseases, including gene knockout models, exogenous gene knock-in models, and site directed mutagenesis models [80], [81].

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Establishing animal models of human diseases

Animal models are crucial tools for understanding gene function, exploring pathogenesis of human diseases and developing new drugs. However, traditional methods for generating animal models are complex, costly and time-consuming, which severely limit the application of animal models in basic medical research and preclinical studies [82]. Since the discovery of CRISPR-Cas systems, a series of genetically modified animal models have successfully been generated in a highly efficient manner [72], [73], [74], [75], [76], [77], [78].

Among numerous model animals, mice are widely used for scientific studies and recognized as the most important model animals in human disease research [83]. So far, researchers have successfully generated many genetically modified mouse models, such as cancer, cardiovascular disease, cardiomyopathy, Huntington's disease, albino, deafness, hemophilia B, obesity, urea cycle disorder and muscular dystrophy [84], [85], [86], [87], [88], [89], [90], [91], [92], [93]. Nevertheless, owing to the great species differences between humans and rodents, they cant provide effective assessment and long-term follow-up for research and treatment of human diseases [94]. Therefore, the application of larger model animals, such as rabbits, pigs and non-human primates, is becoming more and more widespread [74], [77], [78]. With the development of CRISPR-Cas systems, generating larger animal models for human diseases has become a reality, which greatly enriches the disease model resource bank.

Our research focuses on the generation of genetically modified rabbit models using CRISPR-Cas systems. Compared with mice, rabbits are closer to humans in physiology, anatomy and evolution [95]. In addition, rabbits have a short gestation period and less breeding cost. All these make them suitable for studies of the cardiovascular, pulmonary and metabolism diseases [95], [96]. Nowadays, we have generated a series of rabbit models for simulating human diseases, including congenital cataracts, duchenne muscular dystrophy (DMD), X-linked hypophosphatemia (XLH), etc (summarized in ) [97], [98], [99], [100], [101], [102], [103], [104], [105], [106], [107], [108], [109], [110], [111], [112], [113], [114]. Take the generation of PAX4 gene knockout rabbits as an example, the procedure we used to establish genetically modified rabbit models is summarized in and .

CRISPR-Cas system mediated rabbit models of human diseases.

Generation of PAX4 gene knockout (KO) rabbits using CRISPR-Cas9 system. (A) Schematic diagram of the sgRNA target sites located in the rabbit PAX4 locus. PAX4 exons are indicated by yellow rectangles; target sites of the two sgRNA sequences, sgRNA1 and sgRNA2, are highlighted in green; protospacer-adjacent motif (PAM) sequence is highlighted in red. Primers F and R are used for mutation detection in pups. (B) Microinjection and embryo transfer. First a mixture of Cas9 mRNA and sgRNA is microinjected into the cytoplasm of the zygote at the pronuclear stage. Then the injected embryos are transferred into the oviduct of recipient rabbits. After 30days gestation, PAX4 KO rabbits are born. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Summary of the PAX4 KO rabbits generated by CRISPR-Cas9 system.

In addition, the pig is an important model animal extensively used in biomedical research. Compared with mice, their body/organ size, lifespan, anatomy, physiology, metabolic profile and immune characteristics are more similar to those of humans, which makes the pig an ideal model for studying human cardiovascular diseases and xenotransplantation [115]. At present, several genetically modified pig models have been successfully generated, including neurodegenerative diseases, cardiovascular diseases, cancer, immunodeficiency and xenotransplantation model [116], [117], [118], [119], [120], [121], [122].

To date, non-human primates are recognized as the best human disease models. Their advantage is that their genome has 98% homology with the human genome; also, they are highly similar to humans in tissue structure, immunity, physiology and metabolism [123]. Whats more, they can be infected by human specific viruses, which makes them very important models in infectious disease research [124]. Nowadays, researchers have generated many genetically modified monkey models, such as cancer, muscular dystrophy, developmental retardation, adrenal hypoplasia congenita and Oct4-hrGFP knockin monkeys [125], [126], [127], [128], [129].

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Establishing cell models of human diseases

It was found that the efficiency of CRISPR-Cas mediated genome editing is higher in vitro than in vivo, thus the use of genetically modified cell models can greatly shorten the research time in medical research [130]. Until now, researchers have used CRISPR-Cas systems to perform genetic manipulations on various cell lines, such as tumor cells, adult cells and stem cells, in order to simulate a variety of human diseases [79], [80].

Fuchs et al. generated the RPS25-deficient Hela cell line by knocking out ribosomal protein eS25 (RPS25) gene using CRISPR-Cas9 system [131]. Drost et al. edited four common colorectal cancer-related genes (APC, P53, KRAS and SMAD4) in human intestinal stem cells (hISCs) by CRISPR-Cas9 technology [132]. The genetically modified hISCs with 4 gene mutations possessed the biological characteristics of intestinal tumors and could simulate the occurrence of human colorectal cancer [132]. Jiang et al. induced site-specific chromosome translocation in mouse embryonic stem cells by CRISPR-Cas9, in order to establish a cell and animal model for subsequent research on congenital genetic diseases, infertility, and cancer related to chromosomal translocation [133].

In addition, induced pluripotent stem cells (iPSCs) have shown great application prospect in disease model establishment, drug discovery and patient-specific cellular therapy development [134]. iPSCs have the ability of self-renewal and multiple differentiation potential, which are of great significance in disease model establishment and regenerative medicine research [135]. In recent years, by combining CRISPR-Cas systems with iPSC technology, researchers have generated numerous novel and reliable disease models with isogenic backgrounds and provided new solutions for cell replacement therapy and precise therapy in a variety of human diseases, including neurodegenerative diseases, acquired immunodeficiency syndrome (AIDS), -thalassemia, etc [134], [135], [136].

With the development of CRISPR-Cas systems and the discovery of novel Cas enzymes (Cas12, Cas13, etc.), CRISPR-based molecular diagnostic technology is rapidly developing and has been selected as one of the world's top ten science and technology advancements in 2018 [137].

Unlike Cas9, Cas13 enzymes possess a collateral cleavage activity, which can induce cleavage of nearby non-target RNAs after cleavage of target sequence [54]. Based on the collateral cleavage activity of Cas13, Feng Zhang et al.[138] developed a Cas13a-based in vitro nucleic acid detection platform, named SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing). It is composed of Cas13a, sgRNA targeting specific RNA sequences and fluorescent RNA reporters. After Cas13a protein recognizes and cleaves the target RNA, it will cut the report RNA and release the detectable fluorescence signal, so as to achieve the purpose of diagnosis [138]. Researchers have used this method to detect viruses, distinguish pathogenic bacteria, genotype human DNA and identify tumor DNA mutations [137], [138]. Later, Feng Zhang et al. improved SHERLOCK system and renamed it as SHERLOCKv2, which can detect four virus at the same time [139].

In addition to Cas13, Cas12 enzymes are also found to possess collateral cleavage activity [140]. Doudna et al.[141] developed a nucleic acid detection system based on Cas12a (also known as Cpf1), named DETECTR (DNA endonuclease-targeted CRISPR trans reporter). DETECTR has been used to detect cervical cancer associated HPV subtypes (HPV16 and HPV18) in either virus-infected human cell lines or clinical patient samples [141]. Furthermore, Doudna et al. are trying to use the newly discovered Cas14 and CasX proteins in molecular diagnosis, which may further enrich the relevant techniques of CRISPR-based molecular diagnosis [142], [143].

CRISPR-based molecular diagnostic technology has incomparable advantages over traditional molecular diagnostic methods, such as high sensitivity and single-base specificity, which is suitable for early screening of cancer, detection of cancer susceptibility genes and pathogenic genes [137], [144]. Meanwhile, CRISPR diagnostics is inexpensive, simple, fast, without special instrument, and is suitable for field quick detection and detection in less-developed areas [137], [144]. At present, many companies are trying to develop CRISPR diagnostic kits for family use, to detect HIV, rabies, Toxoplasma gondi, etc.

CRISPR-Cas9 system enables genome-wide high-throughput screening, making it a powerful tool for functional genomic screening [145]. The high efficiency of genome editing with CRISPR-Cas9 system makes it possible to edit multiple targets in parallel, thus a mixed cell population with gene mutation can be produced, and the relationship between genotypes and phenotypes could be confirmed by these mutant cells [146]. CRISPR-Cas9 library screening can be divided into two categories: positive selection and negative selection [147]. It has been utilized to identify genes associated with cancer cell survival, drug resistance and virus infection in various models [148], [149], [150]. Compared with RNAi-based screening, high-throughput CRISPR-Cas9 library screening has the advantages of higher transfection efficiency, minimal off-target effects and higher data reproducibility [151]. At present, scientists have constructed human and mouse genome-wide sgRNA libraries, and they have been increasingly improved according to different requirements [152], [153]. In the future, CRISPR-Cas9-based high-throughput screening technology will definitely get unprecedented development and application.

Gene therapy refers to the introduction of foreign genes into target cells to treat specific diseases caused by mutated or defective genes [154]. Target cells of gene therapy are mainly divided into two categories: somatic cells and germ line cells. However, since germ line gene therapy is complicated in technique as well as involves ethical and security issues, today gene therapy is limited to somatic cell gene therapy [155]. Traditional gene therapy is usually carried out by homologous recombination or lentiviral delivery. Nevertheless, the efficiency of homologous recombination is low, and lentiviral vectors are randomly inserted into the recipient genome, which may bring potential security risks to clinical applications [156]. Currently, with the rapid development of CRISPR-Cas systems, they have been widely applied in gene therapy for treating various of human diseases, monogenic diseases, infectious diseases, cancer, etc [155], [156], [157]. Furthermore, some CRISPR-mediated genome-editing therapies have already reached the stage of clinical testing. briefly summarizes the ongoing clinical trials of gene therapy using genome-editing technology, including ZFN, TALEN and CRISPR-Cas systems.

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Monogenic diseases

Monogenic diseases refer to the genetic diseases caused by mutations of a single allele or a pair of alleles on a pair of homologous chromosomes [158]. There are more than 6600 known monogenic diseases around the world, -thalassaemia, sickle cell disease (SCD), hemophilia B (HB), retinitis pigmentosa (RP), leber congenital amaurosis type 10 (LCA10), duchenne muscular dystrophy (DMD), hutchinson-gilford progeria syndrome (HGPS), hereditary tyrosinemia (HT), cystic fibrosis (CF), etc [159]. Most of the monogenic diseases are rare diseases lacking of effective treatment, which will greatly affect the life quality of patients. Nowadays, many animal models of monogenic diseases have been treated with CRISPR-mediated gene therapy. Furthermore, even some CRISPR clinical trials for monogenic diseases are going on [160].

Summary of clinical trials of gene therapy using genome-editing technology.

-Thalassaemia, a hereditary hemolytic anemia disease, is one of the most common and health-threatening monogenic diseases in the world. It is characterized by mutations in the -globin (HBB) gene, leading to severe anemia caused by decreased hemoglobin (Hb) level [161]. For the moment, the only way to cure -thalassemia is hematopoietic stem cell transplantation (HSCT). Yet, high cost of treatment and shortage of donors limit its clinical application [162]. Other therapy, for example, blood transfusion, can only sustain the life of patients but cant cure the disease [161]. To better treat -thalassemia, researchers have turned their attention to gene therapy. A major technical idea is to repair the defective -globin gene of iPSCs from patients with -thalassemia by CRISPR-Cas9 technology, then red blood cells can be produced normally and the disease could be cured [163], [164]. Besides, reactivating fetal hemoglobin (HbF) expression has also been proposed to be an effective method to treat -thalassemia through knockout of BCL11A gene, which suppresses the expression of fetal hemoglobin [165], [166].

Additionally, CRISPR-Cas systems have also been used for the treatment of other hematologic diseases, such as sickle cell disease (SCD) and hemophilia B (HB). SCD is a monogenic disease caused by a single-nucleotide mutation in human -globin gene, leading to a substitution of glutamic acid by valine and the production of an abnormal version of -globin, which is known as hemoglobin S (HbS) [167]. CRISPR-Cas9 system has been used to treat SCD by repairing the -globin gene mutation or reactivating HbF expression [168], [169]. HB is an X-linked hereditary bleeding disorder caused by deficiency of coagulation factor IX, and the most common treatment for hemophilia B is supplement blood coagulation factor [170], [171]. Huai et al. injected naked Cas9-sgRNA plasmid and donor DNA into the adult mice of F9 mutation HB mouse model for gene correction [172]. Meanwhile, Cas9/sgRNA were also microinjected into germline cells of this HB mouse model for gene correction. Both in vivo and ex vivo experiment were sufficient to remit the coagulation deficiency [172]. Guan et al. corrected the F9 Y371D mutation in HB mice using CRISPR-Cas9 mediated in situ genome editing, which greatly improved the hemostatic efficiency and increased the survival of HB mice [173].

Duchenne muscular dystrophy (DMD) is an X-chromosome recessive hereditary disease, with clinical manifestations of muscle weakness or muscle atrophy due to a progressive deterioration of skeletal muscle function [174]. It is usually caused by mutations in the DMD gene, a gene encoding dystrophin protein [174]. Deletions of one or more exons of the DMD gene will result in frameshift mutations or premature termination of translation, thereby normal dystrophin protein can not be synthesized [175]. Currently, there is no effective treatment for DMD. Conventional drug treatment can only control the disease to a certain extent, but can not cure it. It was found that a functional truncated dystrophin protein can be obtained by removing the mutated transcripts with CRISPR-Cas9 system [176], [177], [178]. In addition, base editing systems can also be applied in DMD treatment by repairing single base mutation or inducing exon skipping by introducing premature termination codons (PTCs) [179].

Retinitis pigmentosa (RP) is a group of hereditary retinal degenerative diseases characterized by progressive loss of photoreceptor cells and retinal pigment epithelium (RPE) function [180]. RP has obvious genetic heterogeneity, and the inheritance patterns include autosomal dominant, autosomal recessive, and X-linked recessive inheritance [180]. To date, there is still no cure for RP. In recent years, with the rapid development of gene editing technology, there has been some progress in the treatment of RP. Several gene mutations causing RP have been corrected by CRISPR-Cas9 in mouse models to prevent retinal degeneration and improve visual function, for example, RHO gene, PRPF31 gene and RP1 gene [181], [182].

Leber Congenital Amaurosis type 10 (LCA10) is an autosomal retinal dystrophy with severe vision loss at an early age. The most common gene mutation found in patients with LCA10 is IVS26 mutation in the CEP290 gene, which disrupts the coding sequence by generating an aberrant splice site [183]. Ruan et al. used CRISPR-Cas9 system to knock out the intronic region of the CEP290 gene and restored normal CEP290 expression [184]. In addition, subretinal injection of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing [185], [186].

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare lethal genetic disorder with the characteristic of accelerated aging [187]. A point mutation within exon 11 of lamin A gene activates a cryptic splice site, leading to the production of a truncated lamin A called progerin [188]. However, CRISPR-Cas based gene therapy has opened up a broad prospect in HGPS treatment. Administration of AAV-delivered CRISPR-Cas9 components into HGPS mice can reduce the expression of progerin, thereby improved the health condition and prolonged the lifespan of HGPS mice [189], [190]. In addition, Suzuki et al. repaired G609G mutation in a HGPS mouse model via single homology arm donor mediated intron-targeting gene integration (SATI), which ameliorated aging-associated phenotypes and extended the lifespan of HGPS mice [191].

CRISPR-Cas systems have also showed their advantages in gene therapy of hereditary tyrosinemia (HT) and cystic fibrosis (CF). HT is a disorder of tyrosine metabolism caused by deficiency of fuarylacetoacetate hydrolase (Fah) [192]. Yin et al. corrected a Fah mutation in a HT mouse model by injecting CRISPR-Cas9 components into the liver of the mice [193]. Then, the wild-type Fah protein in the liver cells began to express and the body weight loss phenotype was rescued [193]. CF, an autosomal recessive inherited disease with severe respiratory problems and infections, has a high mortality rate at an early age [194]. It is caused by mutations in the CFTR gene, which encodes an epithelial chloride anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR) [194]. Until now, genome editing strategies have been carried out in cell models to correct CFTR mutations. In cultured intestinal stem cells and induced pluripotent stem cells from cystic fbrosis patients, the CFTR homozygous 508 mutation has been corrected by CRISPR-Cas9 technology, leading to recovery of normal CFTR expression and function in differentiated mature airway epithelial cells and intestinal organoids [195], [196].

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Infectious diseases

In recent years, gene therapy has gradually been applied to the treatment of viral infectious diseases. Transforming host cells to avoid viral infection or preventing viral proliferation and transmission are two main strategies for gene therapy of viral infectious diseases [197].

Human immunodeficiency virus (HIV), a kind of retrovirus, mainly attacks the human immune system, especially the CD4 T lymphocytes. When human cells are invaded by HIV, the viral sequences can be integrated into the host genome, blocking cellular and humoral immunity while causing acquired immunodeficiency syndrome (AIDS) [198]. There is still no known cure for AIDS but it could be treated. Although antiretroviral therapy can inhibit HIV-1 replication, the viral sequences still exist in the host genome, and they could be reactivated at any time [199]. CRISPR-Cas9 system can target long terminal repeat (LTR) and destruct HIV-1 proviruses, thus it is possible to completely eliminate HIV-1 from genome of infected host cells [200], [201]. In addition, resistance to HIV-1 infection could be induced by knockout of the HIV co-receptor CCR5 gene in CD4 T cells [202], [203].

Cervical cancer is the second most common gynecologic malignant tumor. The incidence is increasing year by year and young people are especially prone to this disease. It was found that the occurrence of cervical cancer is closely related to HPV (human papillomavirus) infection [204]. HPV is a double-stranded cyclic DNA virus, E6 and E7 genes located in HPV16 early regions are carcinogenic genes [205]. Researchers designed sgRNAs targeting E6 and E7 genes to block the expression of E6 and E7 protein, subsequently the expression of p53 and pRb was restored to normal, finally increasing tumor cells apoptosis and suppressing subcutaneous tumor growth in in vivo experiments [206], [207], [208]. Moreover, HPV virus proliferation was blocked through cutting off E6/E7 genes, and the virus in the bodies could be eliminated [206], [207], [208].

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Cancer

Cancer is the second leading cause of death worldwide after cardiovascular diseases, and it is also a medical problem that needs to be solved urgently. A variety of genetic or epigenetic mutations have been accumulated in the cancer genome, which can activate proto-oncogenes, inactivate tumor suppressors and produce drug resistance [209], [210]. So far, CRISPR-Cas systems have been used to correct the oncogenic genome/epigenome mutations in tumor cells and animal models, resulting in inhibition of tumor cell growth and promotion of cell apoptosis, thereby inhibiting tumor growth [211], [212], [213].

In addition, immunotherapy is considered to be a major breakthrough in cancer treatment, especially chimeric antigen receptor-T (CAR-T) cell therapy, which has a significantly therapeutic effect on leukemia, lymphoma and certain types of solid tumors [214], [215], [216]. CAR-T cells are genetically manipulated, patient-specific T cells, which express receptors targeting antigens specially expressed on tumor cells, for example, CD19 CAR-T cells for B cell malignancies. Then these cells will be transfused back to patients to fight against cancer [217]. However, CAR-T cell therapy is complex, time-consuming and expensive, and it is greatly limited by the quality and quantity of autologous T cells. Therefore, researchers have used CRISPR-Cas9 system to develop universal CAR-T cells, such as simultaneously removing endogenous T cell receptor gene and HLA class I encoding gene on T cells of healthy donors and introducing CAR sequence [218], [219], [220]. Thereby, it could be used in multiple patients without causing graft versus host reaction (GVHR). In addition, CRISPR-Cas mediated genome editing has also been used to enhance the function of CAR-T cells by knocking out genes encoding signaling molecules or T cell inhibitory receptors, such as programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) [221], [222].

Though CRISPR-Cas mediated efficient genome editing technologies have been broadly applied in a variety of species and different types of cells, there are still some important issues needed to be addressed during the process of application, such as off-target effects, delivery methods, immunogenicity and potential risk of cancer.

It was found that designed sgRNAs will mismatch with non-target DNA sequences and introduce unexpected gene mutations, called off-target effects [223]. Off-target effects seriously restrict the widespread application of CRISPR-Cas mediated genome editing in gene therapy, for it might lead to genomic instability and increase the risk of certain diseases by introducing unwanted mutations at off-target sites [224]. At present, several strategies have been used to predict and detect off-target effects, online prediction software, whole genome sequencing (WGS), genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), discovery of in situ cas off-targets and verification by sequencing (DISCOVER-Seq), etc [225]. Furthermore, to minimize off-target effects, researchers have systematically studied the factors affecting off-target effects and developed a number of effective approaches.

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Rational design and modification of sgRNAs

The specific binding of sgRNA with the target sequence is the key factor in CRISPR-Cas mediated genome editing. Rational design of highly specific sgRNAs might minimize off-target effects [224]. The length and GC content of sgRNAs, and mismatches between sgRNA and its off-target site will all affect the frequency of off-target effects [226]. In addition, on the basis of rational design of sgRNAs, the specificity of CRISPR-Cas systems can be further improved by modifying sgRNAs, such as engineered hairpin sgRNAs and chemical modifications of sgRNAs [227], [228].

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Modification of Cas9 protein

As we know, the interaction between Cas9 and DNA affects the stability of DNA-Cas9/sgRNA complex as well as tolerance to mismatch [229]. Therefore, high-fidelity SpCas9 variants have been developed by introducing amino substitution(s) into Cas9 protein in order to destabilize the function structure of the CRISPR complex [230]. Researchers have developed several highly effective Cas9 mutants, high-fidelity Cas9 (SpCas9-HF1), enhanced specificity Cas9 (eSpCas9), hyper-accurate Cas9 (HypaCas9), etc [231], [232], [233]. All of them can significantly reduce off-target effects while retain robust target cleavage activity.

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Adoption of double nicking strategy

Recently, a double-nicking strategy has been developed to minimize off-target effects, which employs two catalytic mutant Cas9-D10A nickases and a pair of sgRNAs to produce a cleavage on each strand of the target DNA, thus forming a functional double strand break [234]. Additionally, it was proven that the fusion protein generated by combining dCas9 with Fok nuclease can also reduce off-target effects [235]. Only when the two fusion protein monomers are close to each other to form dimers, can they perform the cleavage function [235]. This strategy could greatly reduce DNA cleavage at non-target sites.

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Anti-CRISPRs

Off switches for CRISPR-Cas9 system was first discovered by Pawluk et al. in 2016. They identified three naturally existing protein families, named as anti-CRISPRs, which can specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis[236]. Later, Rauch et al. discovered four unique type IIA CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages, and two of them (AcrllA2 and AcrllA4) can block SpCas9 when assayed in Escherichia coli and human cells [237]. Recently, Doudna et al. discovered two broad-spectrum inhibitors of CRISPR-Cas9 system (AcrllC1 and AcrllC3) [238]. Therefore, in order to reduce off-target effects, the anti-CRISPRs could be used to prevent the continuous expression of Cas9 protein in cells to be edited.

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Others

The concentration of Cas9/sgRNA can also affect the frequency of off-target mutations [239]. Thus, the optimal concentration of Cas9 and sgRNA needs to be determined by pre-experiment. Besides, the formulation of CRISPR-Cas9 can affect the frequency of off-target mutations as well. Cas9 nucleases can be delivered into target cells in 3 different forms: DNA expression plasmid, mRNA or recombination protein [240]. Currently, the use of Cas9/sgRNA ribonucleoprotein complexes (Cas9-RNPs), which are composed of purified Cas9 proteins in combination with sgRNA, is becoming more and more widespread. It was found that delivery as plasmid usually produces more off-targets than delivery as RNPs, since the CRISPR-Cas system is active for a shorter time without Cas9 transcription and translation stages [241], [242].

Nowadays, how to effectively deliver CRISPR-Cas components to specific cells, tissues and organs for precisely directed genome editing is still a major problem in gene therapy. Ideal delivery vectors should have the advantages of non-toxicity, well targeting property, high efficiency, low cost, and biodegradability [35], [156]. At present, three main delivery methods have been employed in delivering CRISPR-Cas components, including physical, viral and non-viral methods [243]. Physical methods are the simplest way to deliver CRISPR-Cas components, including electroporation, microinjection and mechanical cell deformation. They are simple and efficient, which can also improve the expression of genes, and being widely applied in in vitro experiments [243], [244]. In addition, viral vectors, such as adenovirus, adeno-associated virus (AAV) and lentivirus viral vectors, are being widely used for both in vitro/ex vivo and in vivo delivery due to their high delivery efficiency. They are commonly used for gene delivery in gene therapy, and some of them have been approved for clinical use [245], [246]. However, safety issue of viral vectors is still a major problem needed to be solved in pre-clinical trials. Therefore, researchers have turned their attention to non-viral vectors, for instance, liposomes, polymers and nanoparticles [247]. Based on the advantages of safety, availability and cost-effectiveness, they are becoming a hotspot for the delivery of CRISPR-Cas components [248].

Since all these delivery methods have both advantages and disadvantages, its necessary to design a complex of viral vectors and non-viral vectors, which combines the advantages of both vectors. Along with the deepening of research, various carriers could be modified by different methods to increase the delivery efficiency and reduce the toxicity [249]. In addition, more novel vectors, such as graphene and carbon nanomaterials (CNMs), could also be applied in the delivery of CRISPR-Cas components [250], [251].

Since the components of CRISPR-Cas systems are derived from bacteria, host immune response to Cas gene and Cas protein is regarded as one of the most important challenges in the clinical trials of CRISPR-Cas system [156], [252]. It was found that in vivo delivery of CRISPR-Cas components can elicit immune responses against the Cas protein [252], [253]. Furthermore, researchers also found that there were anti-Cas9 antibodies and anti-Cas9 T cells existing in healthy humans, suggesting the pre-existing of humoral and celluar immune responses to Cas9 protein in humans [254]. Therefore, how to detect and reduce the immunogenicity of Cas proteins is a major challenge will be faced in clinical application of CRISPR-Cas systems. Researchers are trying to handle this problem by modifying Cas9 protein or using Cas9 homologues [255].

Recently, two independent research groups found that CRISPR-Cas mediated double-stranded breaks (DSBs) can activate the p53 signaling pathway [256], [257]. This means that genetically edited cells are likely to become potential cancer initiating cells, and clinical treatment with CRISPR-Cas systems might inadvertently increase the risk of cancer [256], [257], [258]. Although there is still no direct evidence to confirm the relationship between CRISPR-Cas mediated genome editing and carcinogenesis, these studies once again give a warning on the application of CRISPR-Cas systems in gene therapy. It reminds us that there is still a long way to go before CRISPR-Cas systems could be successfully applied to humans.

CRISPR-Cas mediated genome editing has attracted much attention since its advent in 2012. In theory, each gene can be edited by CRISPR-Cas systems, even genes in human germ cells [259]. However, germline gene editing is forbidden in many countries including China, for it could have unintended consequences and bring ethical and safety concerns [260].

However, in March 2015, a Chinese scientist, Junjiu Huang, published a paper about gene editing in human tripronuclear zygotes in the journal Protein & Cell, which brings the ethical controversy of human embryo gene editing to a climax [261]. Since then, genome editing has been challenged by ethics and morality, and legal regulation of genome editing has triggered a heated discussion all around the world.

Then, on Nov. 28, 2018, the day before the opening of the second international human genome editing summit, Jiankui He, a Chinese scientist from the Southern University of Science and Technology, announced that a pair of gene-edited babies, named Lulu and Nana, were born healthy in China this month. They are the worlds first gene-edited babies, whose CCR5 gene has been modified, making them naturally resistant to HIV infection after birth [262]. The announcement has provoked shock, even outrage among scientists around the world, causing widespread controversy in the application of genome editing.

The society was shocked by this breaking news, for it involves genome editing in human embryos and propagating into future generations, triggering a chorus of criticism from the scientific community and bringing concerns about ethics and security in the use of genome editing. Therefore, scientists call on Chinese government to investigate the matter fully and establish strict regulations on human genome editing. Global supervisory system is also needed to ensure genome editing of human embryos moving ahead safely and ethically [263].

Since CRISPR-Cas mediated genome editing technologies have provided an accessible and adaptable means to alter, regulate, and visualize genomes, they are thought to be a major milestone for molecular biology in the 21st century. So far, CRISPR-Cas systems have been broadly applied in gene function analysis, human gene therapy, targeted drug development, animal model construction and livestock breeding, which fully prove their great potential for further development. However, there are still some limitations to overcome in the practical applications of CRISPR-Cas systems, and great efforts still need to be made to evaluate their long-term safety and effectiveness.

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