November 2023
Editorial Team
Jon Brudvig, PhD – Editor, The Vector
Jessica Schneller, PhD – Associate Editor, The Vector
Wan Du, MD, PhD – Junior Editor, The Vector
Inside This Issue
Leadership Message
Breaking Through
From Molecular Therapy
Society News
Career Center
Public Policy
Industry News
Leadership Message
#ASGCT24 Abstract Submission Opens Next Week
Hello ASGCT members,
It’s hard to believe it, but abstract submission for the ASGCT 27th Annual Meeting is already upon us! This Monday, Nov. 13, is the first day you can send us your research for possible presentation at the Annual Meeting May 7-11 in Baltimore, MD.
Abstract submission is the best way to get your work in front of the largest community of CGT leaders in one forum. If accepted, you’ll have the opportunity to present your research and talk about its implications at the meeting with your colleagues. Your work will also be published in our flagship Molecular Therapy journal and you might be eligible for awards. If you’re an associate member of ASGCT and a first and presenting abstract author, you qualify for free registration. Take a look at the abstract categories (you even have a few additional abstract categories to choose from this year!) and get ready to submit your abstract through Jan. 26, 2024. New for the 2024 abstract submission period, we’ve added a spot for investigators to include a plain language summary. We look forward to learning from your discoveries next year!
As the Annual Meeting planning continues to take shape, we’re still in renewal season here at ASGCT. Before meeting registration opens next month, make sure your membership is current and renew today. It took me less than five minutes, so please join us in securing your benefits through 2024. Know anyone who’s not a member? They can join now and get free registration for the rest of 2023. We’re proud to have more programming and research funding than ever and our members are the first to know about it. Renew to maintain your exclusive ASGCT benefits.
All of us at ASGCT are thrilled to begin receiving your Annual Meeting research, and we’re grateful for the work you do. Thank you for your membership.
Sincerely,
Isabelle Rivière, PhD
ASGCT Secretary
Breaking Through
Mechanistic modeling explains the production dynamics of recombinant adeno-associated virus with the baculovirus expression vector system
Destro F, Joseph J, Srinivasan P, Kanter JM, Neufeld C, Wolfrum JM, Barone PW, Springs SL, Sinskey AJ, Cecchini S, Kotin RM, Braatz RD
DOI: https://doi.org/10.1016/j.omtm.2023.05.019
Summary by Jessica Lee Schneller, PhD
Recombinant adeno-associated virus (rAAV) is an important delivery vector for in vivo and ex vivo gene therapy, but current manufacturing processes for rAAV often result in low viral yields and a high percentage of empty viral capsids. The baculovirus expression vector system (BEVS) has been favored for production of rAAV due to the high titer and high fraction of filled capsids. With FDA approval of the first rAAV gene therapy produced in the BEVS platform, there is significant interest in optimizing this production platform to reduce the cost of manufacturing, as rAAV therapies can exceed $1 million per dose. In this study by Braatz and colleagues, a mechanistic model is proposed for baculovirus (BV) infection and rAAV production from insect cells. The model takes into account the steps occurring in BV infection of insect cells to investigate the productivity bottlenecks for the BEVS. The fit of the model for existing BEVS compared to literature data, and potential strategies for increasing productivity of the BEVS based on the findings of the model, are discussed.
The proposed model of rAAV production in the BEVS considered the steps occurring in BV infection of insect cells: BV binding, BV transport to the nucleus and replication, release of budded BV, transcription and translation of AAV genes, rAAV capsid formation, Rep protein synthesis, vector genome amplification, and vector genome encapsidation. In the BEVS, the insect cell line from Spodoptera frugiperda (Sf9) is infected with recombinant baculovirus (BV) expressing the genetic elements for producing rAAV (Rep, Cap, and the vector genome template). In the ThreeBac and TwoBac systems, Sf9 cells are coinfected with either three recombinant BVs or two recombinant BVs, respectively, expressing Rep, Cap, and the gene of interest (goi). The authors first considered BV infection dynamics for the ThreeBac and TwoBac systems, considering viral binding inhibition dynamics and BV transport to the nucleus and replication. They validated their proposed model of BV infection by comparing the model predictions with experimental measurements. The model predictions were found to be consistent with the literature for dynamics parameters such as the viral binding kinetic constants, the kinetic constants for BV endocytosis, release into the cytosol and transport to the nucleus, and levels of intracellular and extracellular BV DNA copies during infection.
AAV production dynamics were then considered, through the intermediate steps of transcription and translation of the AAV structural proteins. Common promoters used in BEVS for expressing AAV proteins, such as the non-structural proteins Rep52 and Rep78, are the polh and p10 promoters as well as the IE1 and ΔIE1 promoters. The transcription kinetics for polh and p10 were estimated by fitting the model predictions to experimental measurements, and the polh promoter was found to lead to slightly stronger transcription than the p10 promoter, consistent with the literature.
The model additionally provided a good fit of the total capsid synthesis measurements, and the experimental measurements validated the model prediction that the total capsid production is larger for batches with a larger ratio between the number of capBV particles per cell and the sum of the repBV and goiBV particles per cell. This observation can be explained by the competition between Rep, Cap and the goi for expression.
The model predictions of Rep52 and Rep78 levels in TwoBac and ThreeBac systems were validated using experimental datasets from the literature, and good agreement was observed for both systems. This was particularly interesting because the model parameters for Rep expression were estimated on data collected in the ThreeBac system only, thereby indicating that the dynamics of Rep transcription are equivalent in the two systems.
The model provided a good fit of the filled capsid production at the end of the batch for the ThreeBac system in the vector genome encapsidation step, the final step in rAAV particle formation. Vector genome amplification requires Rep activity, and therefore vector genome amplification cannot start until Rep78 has been expressed. The model was also able to accurately predict the extent of this limitation in the BEVS. The largest rAAV production achieved experimentally was carried out at the highest multiplicity of infection for all three BVs, according to the model and experimental measurements. While the literature attributes the greater rAAV production in TwoBac versus ThreeBac to a larger coinfected cell population, the model suggests that use of the polh promoter for expressing Rep78 in the TwoBac system rather than the weaker ΔIE1 promoter used in the ThreeBac system, accounts for the higher production of rAAV in the TwoBac system.
The model simulation therefore showed that for the TwoBac system, the concentration of free vector genome available for encapsidation is generally much lower than the concentration of empty capsids, and therefore the non-encapsidated vector genome is the limiting step for rAAV formation, up to the point where the encapsidation rate is controlled by the amplification rate. Vector genome amplification is in turn limited by Rep78. Therefore, higher production of rAAV can be achieved by inducing stronger vector genome amplification and stronger Rep expression. Reducing expression of the transgene and of non-essential BV genes can also lead to increased rAAV production by diminishing the competition for Rep and Cap expression. The authors suggest that in future work, the model could be used to design and test in silico different BV constructs with novel promoter cassette arrangements, potentially leading to enhanced rAAV production.
From Molecular Therapy
Read the position description here and submit your CV and cover letter by Jan. 31, 2024.
Active Calls for Papers
Biomanufacturing in gene and cell therapy (deadline extended to Jan. 15, 2024)
RNA and epigenetic editing: A new generation of precision therapeutics
Novel therapeutic targets and biomarker development
Latest MT issues: Check out the most recent issues of these Molecular Therapy family journals:
Society News
Congratulations to the Science Communication Award Recipients
Anna Blakney, PhD, and Kristina Tatiossian, PhD, are the inaugural recipients of ASGCT's Science Communication Award, which recognizes members who do an outstanding job of communicating their work in cell and gene therapy. Learn more about their work on our blog.
Apply Through Jan. 15 to Share Your CGT Expertise with Policymakers
Want to spend a year working with legislators on Capitol Hill? Apply for the Congressional Policy Fellowship through Jan. 15, 2024. Fellows serve alongside congressional staff, bringing scientific rigor and expertise to the office in which they choose to work. Every fellow will attend a two-week orientation organized by AAAS, covering the workings of the federal government and the key issues, institutions, and individuals involved in federal policymaking.
New Name for MT Family Journal: Molecular Therapy Oncology
The first issue of Molecular Therapy Oncology, formerly Molecular Therapy Oncolytics, will publish in March 2024. The expanded scope of Molecular Therapy Oncology will focus on the development and testing of cancer therapies in pre-clinical and clinical settings. Learn more about the change here.
Career Center
Are you looking for a job in the field of gene and cell therapy? Check out the new ASGCT Career Center for great opportunities with industry, government, and academic organizations. Sign up to receive alerts for open jobs in your area.
If you're from a recruiting institution, advertise in the Featured Jobs section to target the 6,000+ audience of The Vector.
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Public Policy
Members Continue Important Conversations from ASGCT’s Policy Summit
A new publication from Daniela Drago, PhD, a member of ASGCT’s Regulatory Affairs Committee and moderator at the event, summarizes the key learnings and takeaways from the Policy Summit. The article reiterates the unparalleled potential for cell and gene therapies to treat disease, while also acknowledging patient access barriers that exist within the current healthcare system. ASGCT appreciates Dr. Drago lending her voice to this important issue, and all attendees for contributing to these conversations. The Summit tackled unique challenges facing this new class of therapeutics with an expert line up of presenters and keynotes.
Changes to NTAP Timelines Could Create Barriers to Care
ASGCT re-submitted comments to the Centers for Medicare & Medicaid Services (CMS) expressing continued concerns about the changes made to the New Technology Add-on Payment (NTAP) in the Fiscal Year 2024 Inpatient Prospective Payment System (IPPS) proposed rule. These changes have the potential to significantly impact the ability of Medicare beneficiaries to access new therapies and create unnecessary bureaucratic barriers to care.
The changes to NTAP include a shift in the Food and Drug Administration (FDA) marketing authorization deadline from July 1 to May 1 which limits the eligibility of products for NTAP consideration. NTAP has historically played a crucial role in ensuring access and appropriate reimbursement for innovative therapies, including the field’s recent experience with CAR T therapies. Comments reiterated the downstream impact on beneficiary access, and to ensure seamless access, ASGCT requested CMS to provide a grace period or waiver for this new requirement during the first year of implementation. ASGCT engaged on this issue earlier this year, and will continue to watch as the 2024 final rule is implemented.
Robust Funding Necessary for Agencies Supporting CGTs
On Oct. 25, after several rounds of voting stretching over three weeks, Republican Congressman Mike Johnson of Louisiana was elected Speaker with unanimous Republican support. The Speaker of the House is responsible for setting the legislative agenda and determining which bills are brought to the floor for a vote. The Speaker also has significant influence over the appropriations process, which involves allocating funds to various government programs and agencies – including those vital to the CGT field such as FDA and the National Institutes of Health (NIH). The Speaker can use their position to prioritize certain programs and agencies over others, which can have a meaningful impact on the enacted federal budget. Johnson has outlined a proposed legislative schedule for his first year as Speaker, though it remains to be seen how much weight those priorities will have in his day-to-day role.
The next budget deadline Congress must meet is Nov. 17, when the CR from earlier this fall expires and federal funding will lapse. A shutdown would be disruptive and damaging to the federal agencies that CGT researchers, trainees, and developers regularly rely on in their work. For that reason, ASGCT urges Congressional leaders in both the House and Senate to move forward on the appropriations process; and we encourage them to pass a budget that includes robust funding for the agencies that support transformative therapies. The Society will continue to monitor developments for our membership.
CBER Request for Information Open Through Nov. 20
CBER has published a request for information (RFI) from stakeholders regarding scientific challenges and opportunities to advance individualized cellular and gene therapies (CGTs). The opportunities and challenges associated with individualized therapies span the entire development pathway; from robust and consistent manufacturing with assurance of product quality; to nonclinical models and tools to characterize safety and activity; to the generation, collection, and assessment of clinical evidence from an individual patient (or a very small number of patients). FDA intends to use the comments to inform potential planning of future town halls, workshops, or discussion papers which could ultimately facilitate the development of additional regulatory science tools, standards, or guidance.
Stakeholders are encouraged to provide open and honest feedback on areas relating to manufacturing, non-clinical, and clinical development of CGTs before the Nov. 20 deadline. The Society plans to respond and will share comments once posted to the registrar.
Industry News
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