Bringing you the latest cutting-edge research and commentary in bioscience.

Cell & Gene Therapy Insights

Cell & Gene Therapy Insights

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Journal

Welcome to the latest issue of Cell and Gene Therapy Insights – the open access, peer-reviewed publication that brings you the latest insight and commentary from leading experts from academia and industry.

Editorial

Process validation for recombinant adeno-associated virus vectors in rare diseases: challenges for MAAs

Spotlight Article

Editorial

Catherine Cancian

Vector Characterization & Validation

In the past decade, advances in biomedical research have led to the development of novel clinical procedures and biological medicinal products for human use such as gene therapy products. Gene therapy vectors offer new opportunities for the treatment of severe disorders or rare diseases that currently have no or limited therapeutic options. These medicinal products are rapidly emerging as new class of licensed products. The recent example of a recombinant adeno-associated virus (rAAV)-based vector expressing therapeutic gene RPE65 to treat an inherited retinal dystrophy authorized as Luxturna® in the USA and Europe shown great promises for gene therapy and marked a pivotal moment for all gene therapies currently in development. Gene therapy vectors are generated through complex bioprocessing and manufacturing procedures. A major challenge for translation of promising clinical development to commercial phase is the process validation in order to consistently achieve high product safety, potency, purity and stability. As development programs for rare diseases involve smaller study populations, and therefore fewer manufacturing runs, it makes it challenging to follow traditional approaches for process and product characterization. Aspects of this challenge are discussed here.

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Interview

Preparing an ex vivo gene therapy process for process validation

Spotlight Article

Interview

John Moscariello

Vector Characterization & Validation

John Moscariello PhD is Senior Director, Viral Vector and Gene Editing Process Development for Juno Therapeutics, a Celgene Company. John joined Celgene in 2018 and serves as the Senior Director of Viral Vector and Gene Editing Process Development. Prior to joining Celgene, John was the Vice President of Process Development at AGC Biologics (formerly CMC Biologics) where his organization was responsible for all cell line development, upstream and downstream process development, analytical and formulation development, and technology transfer and technical manufacturing support. Prior to AGC Biologics, John held director-level positions at Amgen, focused on process development and characterization, clinical and commercial technology transfer and process validation. John has a Ph.D. in chemical and biological engineering from the University of Wisconsin-Madison and a bachelors of chemical engineering from the University of Delaware.

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Advances and shortfalls in AAV vector characterization

Spotlight Article

Interview

Christine Le Bec

Vector Characterization & Validation

Christine Le Bec joined Genethon in 1997 as a scientist and currently heads the CMC Analytical Department. She is responsible for the analytical activities in the characterization and release testing of gene therapy products at early stage development, stability studies, and interface with CMO for method transfer and validation, analytical/QC testing. She also assists in the response to CMC questions from regulatory agencies query. She has a strong expertise in the development and qualification of analytical methods based on biochemical, biophysical and cell based assays to assess identity, potency, impurity profile, and safety. Before joining Genethon, she obtained her PhD in Bio-Organic Chemistry from Université Pierre et Marie Curie (Paris VI) in 1993. She worked as a postdoctoral researcher at Thomas Jefferson University (Philadelphia, US) and then at Institut Pasteur (Paris, France) in the field of synthesis, structural analysis and in vitro evaluation of antisense DNA as therapeutic agents for cancer and AIDS.

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Preparing to navigate European regulatory requirements for gene therapy process validation

Spotlight Article

Interview

Sol Ruiz

Vector Characterization & Validation

Sol Ruiz is Head of Biologics, Biotechnology and Advanced Therapies at the Spanish Medicines Agency. She has a PhD in Biology (Immunology). She is the chair of the BWP (Biologics Working Party) at the EMA (European Medicines Agency) since March 2014. She is also a member of the CAT (Committee for Advanced Therapies) and a co-opted member of the CHMP (Committee for Human Medicinal Products) since 2007. She also participates in several working groups related to biologics and advanced therapies both at the EMA and at the European Pharmacopoeia.

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Building a LVV characterization and process validation strategy

Spotlight Article

Interview

Michael Burnham

Vector Characterization & Validation

Michael Burnham is the Director of the Vector Development department at Tmunity Therapeutics Inc. in Philadelphia. Mike has over 25 years’ of biosafety and bioprocessing expertise, and is currently working to establish Tmunity’s lentiviral vector manufacturing platform. Prior to joining the team, Mike worked at WuXi AppTec, serving in multiple roles in their Process Development, Viral Production/Purification and Viral Clearance departments. In these roles, Mike and his teams worked to develop new methods for virus purification and quantification, and to improve processes for the purification of client-specific biologics and gene therapy programs. Mike successfully developed novel purification strategies for multiple virus types to improve the growth, potency and quality of viruses used in Viral Clearance studies, as well as developing and validating assays for emerging viruses. He holds a BS in Biological Sciences from the University of Delaware and an MS in Biological Sciences from Rutgers University.

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Perspective

Analytical Technology Used in the Latest Development of Gene Therapy Candidates

Spotlight Article

Perspective

Zhenhong Li, Zhuchun Wu, Tomoko Maekawa, Win Den Cheung, Keith Webber, Li Zhi, Kevin O’Brian, Brian Howie, Claire Zhang, Bhargavi Kondragunta, Tristan Marshall, & Franz Gerner

Vector Characterization & Validation

Adeno-associated virus (AAV)-based vectors expressing therapeutic gene products have shown great promise for human gene therapy. In the last 3-5 years, significant resources have been invested in the development and evaluation of these therapies in clinical studies. Commercial products are emerging which provide significant benefit to people with debilitating diseases. These great strides forward in gene therapy are only possible with the support of sophisticated analytical technology. Some of the advancements in analysis of well-characterized biologics can be leveraged to further development in the molecular therapy arena. In addition, new analytical applications are needed to facilitate the accelerated development time lines often encountered in gene therapy and the rare disease space. This article captures the latest developments in analytical technology that provides key measurements and characterization support for the development of gene therapy products at REGENXBIO Inc.

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Expert Insight

Challenges and opportunities of machine-guided capsid engineering for gene therapy

Spotlight Article

Expert Insight

Eric D Kelsic & George M Church

Vector Characterization & Validation

Advances in DNA delivery will be crucial to the enablement of new gene therapies. Pioneering efforts in developing recombinant Adeno-Associated Virus (AAV) technology for gene delivery have led to a recent wave of treatments for genetic diseases with great unmet need. Most current therapies use the capsids of AAV isolated from Adenovirus stocks and primary human and primate tissues, that were then discovered to have favorable tropism, immunogenicity and manufacturability properties. However, despite much work invested in the engineering of new capsids for enhanced delivery, many delivery targets remain out of reach. In our view, high-throughput capsid engineering could be done more effectively by combining three advanced technologies in a closed-loop manner: i. next-gen library synthesis, ii. next-gen sequencing, and iii. machine learning. This approach enables a machine-guided data-driven workflow in which the search for improved capsids is dramatically accelerated relative to traditional open-loop methods. In this report, we review the technological advances that are pushing the field of AAV capsid engineering toward machine-guided methods, describe and explore the promise of this new approach, and discuss anticipated challenges. In the near future, machine-guided approaches will revolutionize our ability to design safe, targeted, delivery tools for the treatment of genetic conditions.

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The ongoing evolution and translational potential of novel AAV vectors for human gene therapy

Spotlight Article

Expert Insight

Sabrina Sun & David V Schaffer

Vector Characterization & Validation

Recombinant adeno-associated viral (AAV) vectors have irrefutably taken the lead in gene therapy clinical trials, gaining overwhelming popularity in the past decade as a result of their ability to safely mediate long-term expression and efficacy. While the FDA’s approval of Luxturna last year and the wave of Phase 1/2 studies coming down the clinical pipeline are strong testaments to the therapeutic promise of AAV-based gene therapy, improvements in delivery efficiency, target specificity and resistance to neutralizing antibodies of AAV vectors in humans are crucial for expanding patient eligibility and adapting the technology to a broad range of diseases. AAV’s minimalistic genome, in which all capsid proteins are encoded by a single gene, has enabled accelerated development of capsid design and evolution strategies, culminating in recent FDA clearance of several novel AAV variants for use in clinical studies. This review describes the latest trends in AAV vector engineering that harness cell-type-specific selection schemes and our rapidly accumulating knowledge of AAV capsid structure and function to overcome longstanding obstacles in the gene therapy field. Next-generation sequencing platforms that have powered unprecedented, high-throughput phenotypic profiling of AAV vectors are also discussed. As these innovations fuel the remarkable momentum in the field, outstanding challenges in scalable manufacturing, immune evasion, and translation of transduction potency across species will shape the ongoing evolution of novel AAV vectors.

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Strategies & technologies for the characterization of adeno-associated virus genomes

Spotlight Article

Expert Insight

Sarah E Sullivan, Emily Menesale, Svetlana Bergelson & Carl Co

Vector Characterization & Validation

Adeno-associated viral (AAV) vectors are being used in a growing number of clinical trials, with many in late phase development. A robust analytical toolkit is essential for ensuring the safety and efficacy of these products. Genome characterization is one of the most critical tools for confirming the identity and quality of AAV products. Methods used for genome characterization vary based on the desired information. For example, alkaline gel electrophoresis allows for visualization of full-length genomes, while agarose gel restriction mapping confirms the fidelity of sequence specific sites across the genome by producing banding patterns stereotypical of the interrogated sequences. Given that AAV is a single-stranded DNA virus, restriction mapping is not straightforward due to the required conversion of the AAV genome to double-stranded DNA prior to enzymatic digestion. Additionally, gel-based readouts are dependent on subjective interpretation by the analyst performing the analysis and this can lead to high variability in reported data. In this paper, we outline strategies for optimizing genome identity characterization via gel-based methods and discuss alternative technologies currently being used for AAV genome analysis that can reduce ambiguity and improve the integrity of viral genome characterization.

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