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Gene Therapy: Current Progress and its Prominence in the Los Angeles Biotech Ecosystem

2024/05/15

Gene Therapy: Current Progress and its Prominence in the Los Angeles Biotech Ecosystem

By Richard Zhuang, Nikki Higa, and Yasaman Moradi

Gene therapy has provided new hope for patients with previously untreatable diseases, offering groundbreaking treatments for conditions such as immune deficiencies, hereditary blindness, hemophilia, beta-thalassemia, and cancer. Over the past two decades, 32 gene therapies have been approved globally, transforming the treatment landscape for many debilitating conditions that were previously incurable or even untreatable. Samir Malhotra, Investment Partner at Civilization Ventures and founding executive of several gene therapy companies, emphasizes that gene therapy is revolutionizing medical treatments by shifting the clinical modality from chronic symptom management to curative, one-time interventions. Consequently, gene therapy is increasingly recognized as the solution to many pressing challenges posed by rare and genetic diseases that are otherwise very challenging to treat, with more than 6000 monogenic diseases afflicting over 350 million patients worldwide (Center for Definitive and Curative Medicine, Stanford University).


The concept of using genetic material as therapeutics has its origins in the notion that genetic material between organisms can be transferred between each other, an idea that led to the birth of the field of genetics. In the 1960s, foundational proof-of-concept experiments in gene therapy were conducted, where scientists demonstrated the possibility of permanently, stably, functionally, and heritably introducing foreign DNA into mammalian cells to establish new genetic functions (Friedmann, 1992). The initial implementation of gene therapy faced significant challenges due to the inefficiency of transferring genetic material into cells. The technological breakthrough for gene transferring efficiency came from utilizing engineered non-pathogenic viruses to carry and deliver the therapeutic genetic cargo, an idea first came out by the end of 1960s (Wirth, et al., 2013).  As Dr. Diana Browning, a staff scientist in the Center for Gene Therapy at City of Hope (Duarte, CA) explains, researchers spent the next two decades exploring various virus types and conducting genetic modifications to achieve functional retroviral vectors that were safe enough to be used on humans. The first approved gene therapy clinical trial in the world was conducted at the Gene Therapy Branch at the National Human Genome Research Institute in 1990, of which they utilized the retroviral vector that were developed in the late 1980s to deliver the therapeutic gene to the extracted patient cells (Wirth, et al., 2013).


Although the first approved gene therapy clinical trial occurred in the US, it was actually the China Food and Drug Administration who approved the first gene therapy in the world in 2003. This first gene therapy was Gendicine, which is an in vivo gene therapy that utilizes a recombinant adenovirus to treat head and neck cancer. (Wirth et al., 2013; Wilson, 2005) Subsequent studies demonstrated Gendicine in combination with chemotherapy and/or radiotherapy has an improved effect compared to conventional therapies alone (Zhang, et al., 2018). 


In recent years, gene therapy has demonstrated significant advancements in both versatility and efficacy, enabling the treatment of a wide range of diseases, including those untreatable by other therapies. This report provides a brief overview of the current applications, and future prospects of gene therapy, with a specific focus on the gene therapy industry in the Los Angeles area.


Current Gene Therapy technology landscape


In Vivo and Ex Vivo Gene Therapies


According to the FDA's 2006 guidance, gene therapy products include nucleic acids, viruses, and genetically engineered microorganisms designed to modify cells either in vivo or ex vivo before administration to the recipient (FDA, 2006; Wirth, et al., 2013). Traditional gene therapies involve delivering therapeutic genes into the target cells to achieve the designed clinical modality. Gene therapies are primarily differentiated by how the genetic material is introduced into the human body. 


In vivo gene therapy: This method involves the direct administration of genetic material into the body via intravenous infusion or targeted organ injection . The primary advantage of in vivo gene therapy is the simplicity and lower relative cost of its administration compared to ex vivo methods. However, in vivo gene therapy faces several challenges, including immune responses to viral vectors, targeted delivery issues, low efficiency of non-viral vectors, potential toxicity to the cells, and often results in only transient therapeutic effects. In vivo gene therapy is mainly used to treat monogenic diseases affecting specific organs (Volodina, et al., 2024).


Ex vivo gene therapy: for this approach, cells are removed from the patient or the donor, genetically modified externally, and reintroduced to the patient’s body. Ex vivo gene therapy allows for the precise cell targeting of cells, and the selection of only the healthy genetically modified cells are re-introduced to the patient’s body reducing potential toxicity. Despite these advantages, ex vivo gene therapy is complex, costly, and limited to certain cell types, with only a small percentage of cells being modifiable. It is predominantly used for treating blood disorders and infectious diseases, such as HIV (Volodina et al., 2024).


A notable advancement in ex vivo gene therapy is the development of CAR T therapy in 2017, which has significantly expanded the applications of gene therapy in cancer that may not be caused by genetic deficiencies (source: NCI). As of now, there are 8 approved CAR T therapies globally, with 6 approved by FDA.


Gene Therapy Delivery Methods


Gene therapy utilizes various delivery methods, each chosen based on specific criteria that include packaging capacity, genome integration, duration of expression, target specificity, and safety (Osborn, 2023). Gene therapy delivery methods can be broadly categorized into three types: viral vector delivery, non-viral vector delivery, and physical methods such as electroporation (Volodina, et al., 2024).


Viral Vectors: Viral vectors are engineered from viruses, leveraging the inherent ability of viruses to efficiently deliver genetic materials. Compared to non-viral vectors, viral vectors are more effective at delivering genetic cargo to target cells. Also, over decades of development, several viral vectors have been well-characterized leading to more predictable outcomes in drug development (Osborn, 2023). However, the use of viral vectors can elicit innate and adaptive immune responses due to their foreign nature. Viral vectors are also the most complex and expensive of gene therapy delivery methods, which results in the high price tag of some of the current gene therapies. Finally, viral vectors exhibit high organism dependent activity, resulting in difficulties to conduct effective preclinical studies. Vectors derived from Adeno-associated viruses (AAV), lentiviruses (a subtype of retrovirus), and other retroviruses are the primary types used, with AAV being the most popular due to its safety profile and effectiveness for in vivo gene therapy. Lentivirus vectors are used mainly in ex vivo gene therapies, while other retrovirus vectors are no longer used in newly developed gene therapies due the potential safety issues (Osborn, 2023; Volodina, et al, 2024). Currently viral vectors are still the most commonly used gene delivery method for gene therapy.


Non-Viral Vectors: Recent advancements have seen increased interest in non-viral vectors such as lipid nanoparticles (LNPs), exosomes, cationic polymers (such as PEI, polysaccharide macromolecules, etc.) and polymer hydrogels, with LNPs and cationic polymeric based vehicles as the most promising ones (Wang, et al., 2023). Compared to viral vectors, non viral vectors have lower cytotoxicity, immunogenicity, and reduced risk of mutagenesis, as well as larger genetic material payload. Current challenges non-viral vesicles face include lower gene transfer efficiency compared to viral vectors, lack of specificity in targeting cells, shorter duration of gene expression, and overall safety concerns (Osborn, 2023; Volodina, et al, 2024). 


Physical methods: Physical methods such as electroporation and microinjection are less common to be applied for gene therapies. These methods can only be utilized for ex vivo gene therapy applications (Volodina et al., 2024). Traditionally this method is mainly used to deliver cargos that are difficult to pack in vesicles, such as the early gene editing modalities like zinc fingers (discussed below) or long genes that surpass available vesicle capacities. However, due to the advancement of the non-viral vectors (discussed in the future outlook section), this delivery method has fallen out of favor due to it being very labor intensive and complicated compared to the other delivery methods available.


Current workflows of gene therapies using different administration and delivery methods are summarized below (Figure 1).


Figure 1

Summary of in vivo and ex vivo gene therapy workflow. Created with BioRender.com


Gene Editing Gene Therapies


Instead of delivering genetic material to the target genome, a newer approach involves the direct editing of the genome in the targeted cells. Gene editing machinery in this case is introduced to the target cell either in vivo or ex vivo (Ho, et al., 2018). Current available genome editing methods include the use of zinc fingers, meganucleases, TALEN, CRISPR genome editing systems, and dCas9 derived genome editors (Li, et al., 2023; Volodina et al., 2024). Casgevy is the only ex vivo gene editing gene therapy that has been approved so far, which utilizes the CRISPR genome editing technology, while no in vivo gene therapies have yet been approved. We will discuss the advantages to apply CRISPR/Cas9 editing technologies in gene therapy in the future outlook session.


Current State of Gene Therapy


Gene Therapy Clinical Trials Overview


Gene therapy has seen a remarkable surge in recent years, a reflection of the advancements in technology. This upward trend is evidenced by the significant increase in the number of gene therapy clinical trials initiated or approved annually since 1989. According to statistics from the Journal of Gene Medicine, as of March 2023, a total of 3,900 clinical trials have been reported globally from 1989 to March 2023. Notably, 1,428 of these trials have occurred from 2017 to March 2023, representing 36.6% of all reported trials in this period (Figure 2A).  Of the 3,900 gene therapy clinical trials examined there is a predominant focus on cancer treatments, which account for 68.3% of all trials (Figure 2B). Beyond the historical prominence of oncology in gene therapy, the intense focus on cancer treatment within this field has been significantly amplified by the development of CAR T therapy since 2017, which has generated substantial interest due to its potential for selectively killing cancer cells.


Together, these statistics underscore the growing momentum of gene therapy as a pivotal strategy for treating a wide spectrum of diseases, with a significant emphasis on oncology.


Figure 2

Clinical trials statistics from all of the 3900 reported global approved/initiated clinical trials as of March, 2023. A) Approved/initiated gene therapy clinical trials globally breakdown by years. B) Indications addressed by gene therapy clinical trials breakdown. Source: The Journal of Gene Medicine, Wiley and Sons


Global Currently Approved Gene Therapies

 

As of April 2024, there are 32 gene therapies currently approved globally. A detailed listing of these therapies is below (modified from BCC Research: BIO225B (2023) and Chancellor et al., 2023).


Table 1: Global Currently Approved Gene Therapies


Currently, 30 out of the 32 approved gene therapies utilize viral vectors as their primary delivery method, highlighting the dominance of viral vector delivery in the field. The distribution between in vivo and ex vivo gene therapies is evenly split, with 16 each, reflecting a balanced advancement in both delivery approaches.


Mirroring the trends observed in gene therapy clinical trials, there has been a noticeable increase in the number of gene therapies approved annually since 2017 (Figure 3A). This growth aligns with the broader trend of accelerated gene therapy development over the past decade. Like in the clinical trials, cancer treatment comprises about half of the currently approved gene therapies, underscoring its predominance in gene therapy applications. Blood genetic diseases, such as hemophilia and sickle cell disease, represent the second most common treatment target, accounting for approximately 20% of approved gene therapies. Neurological genetic disorders are the third most common focus, with about 12.5% of gene therapies aimed at treating these conditions (Figure 3B).

 

Figure 3

Statistics of the 32 gene therapies approved globally as of April, 2024. A) Amount of gene therapies approved globally breakdown by year. B) Treatment target breakdown among the 32 gene therapies have been approved globally. Adopted from  BCC Research: BIO225B (2023) and Chancellor et al., 2023.

 

Current state of the drug pipeline under development

                                               

According to the global research and development database Pharmaprojects, as of June, 2023, the gene therapy drug pipeline comprises 2,070 active programs. Within the current gene therapy drug pipeline, gene-modified cell therapies are the most prevalent, with 1,150 active programs representing 56% of all gene therapies. The second most significant category are gene therapies that involve the transfer of new genetic material, accounting for 719 active programs or 35% of the total. In vivo gene editors, a newer and emerging class, constitute 201 active programs, making up 10% of the pipeline. Approximately one-third of these programs are in the clinical stage, with the remainder in the preclinical phase (Chancellor, et al., 2023).


Oncology treatment is still the majority of the gene therapy pipeline under development, comprising 1,776 of all 2,233 current gene therapy clinical trials (80%). This focus is largely due to the predominance of ex vivo gene therapies, particularly CAR T therapies targeting Hematologic cancers. It is worth noting that ex vivo gene therapy treatments under development targeting non-cancer diseases are exceedingly rare. For in vivo gene therapies under development, the most popular disease target is also oncology, followed by CNS, metabolic and endocrinology, and cardiovascular conditions. Ophthalmology, particularly rare inherited retinal disorders, is another significant area of clinical investigation and falls under the “Other” category. (Chancellor, et al., 2023)


In terms of delivery methods, 49% of the gene therapies currently under development use viral vectors, 5% use non-viral vectors, and 46% have undisclosed delivery methods. AAVs are used in 24% of the current under-development gene therapy pipelines, which is significantly more than other vectors for in vivo gene therapies such as adenovirus (4%), retrovirus (2%), and HSV (1%). Lentivirus vectors took up 11% of the overall gene therapy pipelines (Chancellor, et al., 2023).


Current Challenges in pricing


High costs are a significant challenge for gene therapies. For instance, Orchard Therapeutics’ newly approved one-time gene therapy to treat Early-onset Metachromatic Leukodystrophy, Lenmeldy, is reported to have a wholesale cost of $4.25 million, making it the world’s most expensive drug (Smith, 2024). Gene and cell therapies typically come with high price tags, often exceeding $1 million, far surpassing the median annual cost for other new specialty medicines, which was nearly $30,000 in the U.S. in 2022 (IQVIA, 2024).


A major reason for the high tags is the substantial investment required during the development phase, as implementing novel technologies carries significant risks. If a technology fails the financial loss can be immense, driving up overall costs for the medicines that do succeed. Mr. Malhotra highlights the need for de-risking strategies in gene therapy development to lower production costs and, consequently, prices. Dr. Nicholas Flytzanis, co-founder and Chief Research and Innovation Officer at Capsida Biotherapeutics, notes that while the pricing model for certain gene therapies, which can provide a one-and-done disease modifying treatment, is always going to be different from traditional repeat therapies the current lack of reimbursement incentives is also partly due to the limited number of approved gene therapies. However, as gene therapy becomes more mainstream and the number of approved therapies increases, there will likely be greater incentives to address reimbursement issues, ultimately making these treatments more accessible


Gene Therapy in Los Angeles


The Greater Los Angeles area is home to over 35 gene therapy companies and institutions (Figure 4). Here, we highlight just some of the many exciting members of this community that are driving innovation across the field.


Figure 4

Current gene therapy ecosystem within the Greater Los Angeles area.


Los Angeles Gene Therapy Industry Landscape


Currently, the industry landscape is dominated by companies developing genetically modified cell therapies. Those focused on autologous or patient-derived treatments include Immix Biopharma, ImmPACT Bio, and Neogene Therapeutics (an AstraZeneca company). Immix Biopharma was founded in 2012 and is developing CAR T therapies for AL amyloidosis and autoimmune diseases. Their lead candidate, a BCMA-targeted CAR T, was awarded Orphan Drug Designation by the FDA in 2023 for relapsed/refractory AL amyloidosis and multiple myeloma, and is currently being evaluated in a Phase 1b/2a clinical trial (Immix Biopharm, 2024). ImmPACT Bio, headquartered in West Hills, CA, is focused on a logic-gate-based CAR T platform for the treatment of cancer and autoimmune disease. Earlier this year, they received a $8M California Institute for Regenerative Medicine (CIRM) grant to support a Phase 1b/2 trial of their CD19/CD20-targeted CAR T therapy in refractory lupus nephritis and systemic lupus erythematosus, becoming the first bi-specific CAR T in clinical development for lupus (ImmPACT Bio, 2024). Neogene Therapeutics was founded in 2018 by industry veterans from the CAR T world and quickly became pioneers in developing T-cell receptor (TCR) therapeutics for solid tumors. They were acquired by AstraZeneca in January 2023 for $200M upfront (plus up to an additional $120M) and continue to operate as a subsidiary with locations in Santa Monica and Amsterdam (AstraZeneca, 2023).


Other companies such as Appia Bio and Atara Bio are working on allogeneic or “off the shelf” approaches in which donor-derived cells are genetically engineered. Appia Bio was co-founded in 2020 by David Baltimore based on the work of UCLA investigator, Lili Yang. Appia’s ACUA platform combines hematopoietic stem cell differentiation with CAR gene engineering to produce CAR-NKT cells in a highly scalable manner. In 2021, the company raised $52M in a Series A venture funding round that included 8VC and Two Sigma Ventures (Appia Bio, 2021). Atara Bio started as a spin out of Amgen in 2012 and is one of only a few LA gene therapy companies that have gone public. Their Epstein-Barr virus (EBV) T cell platform enriches donor-derived T cells with TCRs that target EBV and can be engineered to include CARs against additional antigens. The company’s EBV CD19 CAR T candidate is currently in a Phase I trial for B cell non-Hodgkin lymphoma, with another Phase I trial for lupus nephritis expected to begin later this year (Atara Biotherapeutics, 2024).


Meanwhile, companies like A2 Biotherapeutics and Kite Pharma (a Gilead company) have chosen to pursue candidates across both strategies. A2 Biotherapeutics is located in Agoura Hills. Their logic-gated CAR T platform utilizes a two receptor system to activate or block T cell killing depending on the presence of tumor and non-tumor antigens. Their lead candidate is an autologous CEA-targeted CAR T therapy that is currently being investigated in a Phase I/II study for metastatic colorectal, non-small cell lung, and pancreatic cancers (A2 Biotherapeutics, 2024). They have another autologous candidate for solid tumors as well as three allogeneic candidates for undisclosed targets in their pipeline. Kite Pharma was founded in 2009 and was one of the early pioneers in CAR T cell therapies. The company was acquired by Gilead Sciences in 2017 for approximately $11.9B (Gilead Sciences, Inc., 2017). Two of their autologous CD19-targeted CAR T therapies, Yescarta (B-cell lymphoma and Follicular lymphoma) and Tecartus (Mantle cell lymphoma), are amongst the six FDA approved CAR T therapies on the market (Gilead Sciences, Inc., 2024).


In addition to genetically modified cell therapies, AAV-mediated gene therapy products are also being developed by Capsida Biotherapeutics and Oculogenex. Capsida Biotherapeutics was started in 2019 based on an AAV capsid screening platform developed in the lab of Caltech professor, Dr. Viviana Gradinaru. The novel platform identifies engineered capsids that more specifically and efficiently target tissues of interest. The company has since grown to be a full end-to-end provider of AAV gene therapy solutions, a feat that Dr. Flytzanis credits in part to connections with manufacturing expertise from other pharmaceutical groups throughout LA. Capsida currently has three wholly-owned candidates for CNS diseases, plus multiple partnered programs with Abbvie, CRISPR Therapeutics, and Lilly/Prevail Therapeutics (Capsida Biotherapeutics, 2024. Oculogenex was founded in 2020 by ophthalmologist, Hema Ramkumar, with the goal of treating ocular disorders. They are developing AAV-mediated gene therapies that restore expression of epigenetic regulators associated with preventing or slowing retinal and/or macular degeneration (Ramkumar, H.L., 2023).


Lastly, companies specializing in gene therapy manufacturing include Theragent and FUJIFILM Diosynth Biotechnologies. Theragent is CDMO focused on cell and gene therapies. The company was founded to address manufacturing roadblocks in order to accelerate research and clinical trials for the approval of these advanced therapeutics. They currently have two locations, the Covina Discovery labs which houses their Process & Analytical Science development center, and a facility in Arcadia which features 11,250 square feet of manufacturing space plus cleanrooms and support areas (Theragent, 2024). FUJIFILM Diosynth Biotechnologies is a global CDMO with expertise in developing and manufacturing advanced therapies. In 2022, the company took over a 90,000 square-foot cell therapy manufacturing facility in Thousand Oaks, CA for $100M (FUJIFILM, 2022). The acquisition marked FUJIFILM’s first manufacturing site on the West Coast.


Los Angeles Gene Therapy Research Landscape


When discussing the key aspects of gene therapy companies that attract venture capital interest, Mr. Malhotra emphasized the importance of acquiring top-tier talent to build a strong team. Throughout Los Angeles, numerous academic and research centers provide scientific expertise and clinical programs that complement the growing gene therapy industry. Dr. Flytzanis mentioned that the wealth of top talent in preclinical gene therapy research, primarily from the many translational academic labs in LA, influenced their decision to establish their company in the area. Here, we highlight three notable gene therapy initiatives from academic institutes in Los Angeles.


The Center for Gene Therapy at City of Hope focuses on developing stem cell-based therapies to treat cancer, diabetes, AIDS, and other diseases (City of Hope, 2024). The center is dedicated to converting translational research into practical treatments for diseases that are currently incurable. The center collaborates with the City of Hope Alpha Stem Cell Clinic, part of the California Institute for Regenerative Medicine's (CIRMs) Alpha Clinics network, to accelerate the delivery of these life-saving therapies.


The Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research (BSCRC) at UCLA aims to revolutionize disease treatment through personalized cellular therapies and regenerative medicine, where they have conducted in depth research related to how to apply gene therapy in these fields (UC Regents, 2024). Established in 2005, the center fosters interdisciplinary collaboration among faculty members to advance translational research from the laboratory to the clinic. The center has made significant progress in cell and gene therapy including conducting clinical trials to treat various diseases such as immune diseases, and late-stage cancers. BSCRC is also committed to fostering the next generation of scientists in the field of cell and gene therapy translational research. Through the multiple training programs, they currently have 359 trainees, 94 center faculty mentors and more than 250 peer reviewed scientific publications.


The USC/CHLA Cell Therapy Program (CTP), established in 2021, aims to be a world-leading hub for cell and gene therapy, particularly for underserved populations (University of Southern California, 2024). As part of the CIRM alpha clinics network, the program offers comprehensive support to facilitate the translation of academic research into clinical practice, including business strategic planning, product manufacturing, quality control, regulatory compliance, and guidance for FDA investigational new drug (IND) application preparation and submission. Their goal is to help advance cell and gene therapies from preclinical development to clinical trials. Additionally, CTP hosts monthly seminars and classes to educate on drug development and the translation of basic science research into clinical applications with a particular focus on the cell and gene therapy field .


One of the unique resources available to gene therapy companies and academic institutions throughout Los Angeles is the California Institute for Regenerative Medicine or CIRM. The institute was created in 2004 with $3B in funding plus another $5.5B in 2020 to support regenerative medicine research throughout California. In fiscal year 2023-24, CIRM’s $468.3M budget included $84.7M for discovery research, $84.6M towards translational research, $252M for clinical research, $62.5M for infrastructure programs, and $2.5M for educational programs (California Institute for Regenerative Medicine, 2023). Dr. Browning emphasized the pivotal role of CIRM grants, noting that they have been instrumental in bridging funding gaps, particularly for projects at the translational stage.


Gene Therapy Outlook in Los Angeles


Altogether, there are many exciting developments in the LA gene therapy ecosystem. Multiple companies now have promising candidates in clinical trials. Innovations from academic labs throughout the region continue to produce new start-ups. Some of these early-stage companies, including CDR3 Therapeutics and Pluto Immunotherapeutics, are also being supported by local accelerators and incubators like Magnify at the UCLA California NanoSystems Institute.


Looking ahead, future advancement of the gene therapy ecosystem in Los Angeles could greatly benefit from the help of large pharma companies. Dr. Flytzanis envisions that reaching a “critical mass” of gene therapy companies will be key to attracting significant investment and programs such as Eli Lilly’s Gateway Labs for the region. With the combined efforts of stakeholders throughout the gene therapy landscape, LA could solidify its position as a leading hub for gene therapy innovation.


Future Outlooks


Gene therapy stands as a dynamic field in pharmaceutical development, characterized by significant advancements and promising future directions. Here, we mainly focus on two pivotal trends: the enhancement of delivery methods and the expansion of genome editing technologies. 


Development of Delivery Methods


Viral vector delivery is currently the standard in gene therapy and is continuing to see significant new developments. Dr. Browning highlighted that for retroviral vectors such as lentivirus, predominantly used for ex vivo gene therapies, future research will focus on enhancing safety through further genomic engineering and optimizing cell culture conditions to boost transduction efficiency.


Dr. Browning pointed out that the top two types of viral vectors being researched for are lentivirus and AAV, and both have made significant improvement. For AAVs specifically, a major area of innovation is in the optimization for their in vivo gene therapy applications. Current research is concentrated on various engineering strategies to enhance AAVs' transduction efficiency and tissue specificity. This includes AAV capsid engineering to improve vector entry and targeting, as well as the engineering of the cis-regulatory components of the rAAV genome to fine-tune transgene expression in targeted cells (Wang et al., 2024).


Although few non-viral vector-based gene therapies have been developed, there is rapid evolution in this area, such as in lipid-like nanoparticles (LNPs), lipid-like-molecules, exosomes, and a new-generation of cation polymers similar to the the traditionally used PEI, to achieve better performance compared to viral vectors in aspects like application safety, tissue/cell specificity, and cargo compatibility (Wang, et al., 2023). Mr. Malhotra emphasized that non-viral vectors, though still in early development stages, hold potential to significantly broaden the scope of gene therapy. He highlighted the capability of non-viral vectors for carrying large multi-kb genetic cargo, as he believes the next generation of gene therapies is expected to conduct multi-kb level gene insertion for disease treatment, which is beyond the loading capacity of the current viral vectors. Dr. Browning also agreed that non-viral vectors had made considerable progress recently, and will be an important component for the next generation of gene therapies. One important component for next generation gene therapies is in the design of virus-like LNPs (VLPs), a kind of self-assembling protein-based capsular nanoparticles (Kim et al., 2023). Dr. Browning suggested that these particles retain a lot of advantages from the viral vector delivery method, such as high transfection efficiency and potential capability for tissue specific gene cargo delivery, while benefiting from being a non-viral vector. These VLPs are favored for gene editing because their larger packaging capacity facilitates the delivery of essential components like gRNA and endonuclease machinery. 


Both Mr. Malhotra and Dr. Browning agreed that there are benefits to both viral and non-viral vector deliveries, and there is potential for both types to be utilized in future gene therapies. Dr. Browning further suggested that an improved understanding of how lipids influence delivery efficiency could benefit both delivery methods. Since many viral delivery systems incorporate a lipid membrane and non-viral vectors like LNPs and exosomes also use lipids as part of their structure, exploring how lipids affects vesicle properties such as transduction efficiency and tissue/cell-type delivery specificity could identify areas of improvement for the in vivo delivery effectiveness of gene therapies.


Implementation of CRISPR/Cas Technology


The implementation of CRISPR genome editing technology represents a groundbreaking trend in gene therapy, transforming the landscape of human disease treatment. Over the years, CRISPR/Cas has evolved into a versatile toolkit encompassing single-base gene editing, long kb gene insertion, transcriptional regulation, and RNA strand cutting techniques. Continuous advancements have been made in refining these tools, including the development of dead Cas9 (dCas9)—an engineered variant of Cas9 that does not cut DNA but instead directs proteins to specific genomic loci. These innovations have opened doors to a plethora of novel base editing strategies and applications (Li, et al., 2023).


Ralph Valentine Crisostomo, a PhD student from Dr. Donald Kohn's lab at UCLA, told us that CRISPR related technology is currently the hottest topic discussed at the annual meeting of the American Society of Gene & Cell Therapy, held May 7-11 this year (2024) in Baltimore. Mr. Crisostomo’s current research focuses on utilizing the CRISPR/Cas9 system for site-specific insertion of large therapeutic genes to the patient’s genome to treat genetic diseases that are hard to treat currently. He emphasized that CRISPR/Cas9 technology is crucial for enabling insertion of multi-kilobase sequences into a patient’s genome at a target endogenous promoter, as the Cas protein can be guided to specific genome loci with the designed gRNA and the proper PAM sequence. This method significantly reduces cellular toxicity compared to traditional random gene insertion via viral vectors, proving particularly advantageous for in vivo gene therapies. The precise editing facilitated by CRISPR/Cas9 and CRISPR/dCas9 is enabling the creation of innovative gene therapy approaches, including those Mr. Crisostomo is pioneering.


Dr. Browning also expressed enthusiasm for CRISPR's role in gene therapy. She noted an additional benefit of CRISPR/Cas and dCas9: their utility in large-scale screening to identify optimal editing or insertion sites, which can be easily adjusted by modifying the guide RNA. Beside the approach Mr. Crisostomo applied in his research, this side-specific editing capability also makes it possible for targeting intergenic regions to treat diseases, potentially revolutionizing gene therapy's therapeutic reach. Furthermore, Dr. Browning highlighted ongoing efforts to integrate dCas9 into viral vectors, combining viral delivery advantages with reduced cellular toxicity through precise gene insertion. This approach represents a significant step forward in developing the next generation of viral vectors for gene therapy.


Current challenges CRISPR/Cas9 technology faces include its reliance on double-strand breaks (DSBs) that may result in unintended consequences such as random mutations that occur when these breaks are repaired. In contrast, subsequently engineered CRISPR/dCas9 base editing systems avoid intentional generation of DSBs, but can only edit one base/mutation limiting their ability to treat many genetic diseases, as pointed out by Mr. Crisostomo. Mr. Crisostomo mentioned that efforts are underway to develop the next generation of gene editing technology that avoids inducing DSBs while retaining the versatile gene editing function of the CRISPR/Cas9 system, which will further revolutionize the field of gene therapy.


Conclusion


Gene therapy, while rooted in concepts developed decades ago, has recently entered a period of rapid and significant advancement due to decades of development in gene technologies. The evidence presented underscores a trajectory of accelerated growth, suggesting that the field is on the cusp of an exponential expansion. Future iterations of gene therapy are poised to transcend their current scope—extending beyond the treatment of ultra-rare genetic disorders to address more prevalent diseases affecting larger populations. This evolution will likely usher gene therapy into the mainstream of medical practice, promising substantial improvements in human health. We are optimistic that the continued innovation and application of gene therapy will bring transformative benefits to healthcare worldwide.


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