Gene therapy holds enormous promise, revolutionizing the way we approach and treat diseases, including many that were previously considered incurable. The global trend for expansion continues, with more than a thousand cell and gene therapy (CGT) clinical trials underway in 2019.1 Whether it is an introduction of genetic material into target cells in vivo or ex vivo (cell therapy), Adeno-Associated Viruses (AAV) have strengthened their position as a leading platform for gene delivery especially after recent landmark approvals: Luxturna (voretigene neparvovec), an AAV-based therapy that delivers a functional copy of the RPE65 gene to patients with vision loss due to mutation-dependent retinal dystrophy, and Zolgensma (onasemnogene abeparvovec-xioi), which harnesses an AAV (serotype 9) vector to deliver a functional copy of the SMN1 gene to motor neurons in spinal muscular atrophy patients. The pipeline for AAV vector-based therapy is also increasing, with at least 250 clinical trials conducted during 2019.2
AAV vectors are promising gene delivery vehicles because they have an excellent safety profile (they rarely integrate into the host genome). They can transfect both dividing and non-dividing cells, have broad tissue tropisms, the ability to transduce multiple species, and achieve sustained and high-level expression. However, AAV vectors have limited packaging capacity (4.7 kbp), and despite being the least immunogenic therapeutic viral vector, AAV can evoke anti-drug antibodies (ADAs), which may be either pre-existing or developed after the onset of treatment, and this can limit effective gene transfer and nullify transgene expression. Several approaches to AAV vector engineering offer solutions to its limitations and also improve its potential as a gene delivery platform; for example, the inverted terminal repeats (ITRs) can be modified to make them self-complementary, increasing transduction efficiency, and the transgene can be codon-optimized for better protein expression. Both approaches reduce the host anti-AAV immune response.3
As companies transform a gene therapy idea theorized in a lab, from bench to bedside, timely filing of an Investigational New Drug application is the first vital milestone. Here we discuss how to design in vitro and in vivo studies effectively and highlight common “roadblocks” that might delay the Investigational New Drug (IND) submission, strategies to overcome them and focus on specific considerations for AAV-based therapy. Current regulatory guidelines and the expectations of regulatory agencies will be emphasized throughout.
Early Phase Product Development – What to Consider?
Essential questions in early-stage product development are capsid/serotype and expression cassette selection and the impact these decisions might have on manufacturing and scale-up of the product. The choice of AAV serotype is crucial; both tissue tropism and clinical endpoints must be considered. Typically, capsid/serotype selection is based on effective gene delivery in preclinical animal models. However, these do not always accurately predict the human outcome. The use of chimeric (human/mouse) models can help to overcome the species-based discordance in gene transfer and better predict clinical relevance.5 Pre-existing human immunity can also influence the choice of AAV serotype. The prevalence of different anti-AAV neutralizing antibodies (Figure 1) varies according to geographical region.
Therefore, if there is intention to work with a novel AAV serotype, ensure that a human population sample is screened for pre-existing immunity. Our experience indicated that a significant hurdle for assay development and validation was sample availability. Carefully assess how to obtain representative human serum and plasma samples if this type of screening is required. If multiple AAV serotypes are being screened, then the number of samples required rapidly becomes burdensome. A representative pooled sample could be used if the pool does not contain significant outliers.
Each AAV serotype has a particular tissue tropism, leading to AAVspecific biodistribution. AAV-2, for example, does not readily transduce the liver. However, AAV-9 has a broad tissue tropism, including reproductive organs, which could raise safety concerns regarding vertical transmission. The systemic injection of AAV provokes a more significant immune response than other administration routes and there are serotype-specific differences in clearance. AAV-9 persists in the circulation longer than other serotypes.6 Viral scale-up is also serotype dependent. AAV-6, for example, has a poor overall yield that is at least four-fold lower than AAV-9.7
Expression cassettes have multiple components that must be optimized for high and sustained protein expression. Promoters drive transgene expression and can be selected to dictate the location (ubiquitous versus tissue-specific promoters) and temporal (regulatable promoters) expression of target proteins. Ubiquitous promoters produce high levels of target protein, but this may provoke a robust host immune response, limiting gene expression.8 Tissue-specific promoters are physiologically relevant, allow systemic administration and may induce immune tolerance.9 Large promoters such as CBA or CAG can account for up to 36% of the AAV total packaging capacity, potentially limiting the size of the transgene. Other components of an expression cassette, such as poly-A, other regulatory elements, enhancers, or introns should also be optimized, as they can have a direct impact on protein stability and expression. The transgene may be a native, foreign, or chimeric protein and will have a significant impact on the overall immunogenicity of the vector. The biology of the expressed protein is also fundamental. For example, a growth factor might be oncogenic if expressed at a supraphysiological level. Also, performing in vivo and in vitro assays in tandem is critical for product design because there are instances when in vitro assays do not accurately predict in vivo responses.10
Chemistry, Manufacturing and Control (CMC) Assays
To set out some of the potential design challenges and to illustrate how the final product design influences our approach to these assays, we will examine the design and characterization of a critical CMC assay, the in vitro potency assay, which is an absolute requirement for clinical lot release. According to the compliance guidelines, these assays must be conducted in vitro for the biologics license application (BLA) submission.
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The cell line chosen for an in vitro potency assay must be permissive for the selected AAV serotype and promoter, biologically relevant for the target gene so the mechanism of action can be evaluated, have low endogenous target protein expression, and have good growth characteristics. These complex choices are illustrated in the following case study. In rhodopsin-dependent retinitis pigmentosa, the afunctional rhodopsin protein can have associated toxicity. Cideciyan et. al., has developed a product with dual function: an AAV- 5 vector overexpressing functional rhodopsin and shRNA designed to silence mutant endogenous rhodopsin proteins.11 Designing an in vitro potency assay for this vector is demanding since one of the genes of interest is an ion channel with complex biology. AAV-5 intrinsically has a low in vitro transduction efficacy, and the promoter is tissue-specific. Therefore, an AAV-5 permissive retinal cell line that allows monitoring of rhodopsin’s mechanism of action is required to develop an in vitro potency assay. Theoretically, such a cell line can be developed, but the timeline should be considered to keep the end goal in mind. Similarly, some therapeutic genes like human RPGR (retinitis pigmentosa GTPase regulator), which has been a target to treat XLRP (X-linked retinitis pigmentosa), may pose some unique challenges when it comes to developing a potency assay, as RPGR has no well-known biological mechanism of action that can be used to develop an in vitro potency assay.
Not just a potency assay, but the development and validation timeline of other CMC assays like an infectivity assay should also be considered, especially in light of the lack of published guidelines. Should primers/probes be designed for generic (e.g., ITRs) or target-specific sequences? Should techniques like ddPCR or qPCR be used? Should only assay formats like TCID50 or transduction be employed? All these questions, when not answered well in advance, can impact the timeline for IND submission. Therefore, we advise meeting “early and often” with regulatory agencies to determine what is appropriate for individual products.
There is no well-defined set of best practices for developing and manufacturing an in vivo gene therapy. The Alliance for Regenerative Medicine has brought together more than 50 experts with the aim of providing a central standard for design, development, and scalable manufacturing strategies for gene therapy. The proposed document (Project A-Gene) uses an AAV gene therapy case study to illustrate how these strategies can be implemented, and it is scheduled for release in summer 2020.11
AAV can be manufactured and scaled by mainly three different methods (Figure 2), which have inherent advantages and disadvantages. For example, helper virus-based methods are advantageous in that they are suspension cultures that facilitate scale-up. However, the final product may be contaminated with the helper virus. Ensure that appropriate planning occurs for the manufacturing modality of choice.
Intellectual property might not be an obvious concern in early-stage planning. Still, it is vital to determine whether any proposed vector components are subject to any licensing restrictions. The Regenex Bio NAV technology platform holds exclusive rights13 to multiple AAV serotypes, including AAV-9. Failure to recognize this could result in substantial licensing costs in the future. Similarly, promoter- or codon-optimized transgenes could be patented. Cell lines that form the basis for commercial release assays and technology like ddPCR may also have restrictions or licensing issues.
Designing Preclinical Studies to Enable IND Application
Pharmacology studies provide proof of concept data for expression/activity of the protein of interest over time, preliminary information about the optimal route of administration, dosing, and evaluate the efficacy of both the vector and transgene. The FDA recommends selecting an animal species that demonstrates a biological response similar to humans. Some of the critical factors to choose an appropriate animals model are: does the animal model allow vector transduction, is the gene of interest pharmacologically active in the model, is there pre-existing immunity, and could the transgene trigger an immune response in the selected species?
Toxicology studies assess product safety and support proposed clinical investigations. Humoral and cellular immunotoxicity, genotoxicity (genome integration), and reproductive toxicity (e.g., vertical transmission of AAV virus in germ cells) must be assessed. Shedding studies assess horizontal transmission. The FDA requires that all safety studies should be GLP compliant; however, if any toxicity studies are non-GLP, ensure that you include evidence to support why this decision was taken, what other complementary studies were performed under GLP, define the quality system used, how the data was recorded and archived, and include all of this information in the IND package.
Optimizing Regulatory Interactions to Ensure a Successful IND Application
The initial meeting with the FDA is called an initial targeted engagement for regulatory advice on CBER products (INTERACT) meeting. Its primary goal is an early discussion before the pre-IND meeting and serves as an opportunity to discuss in-vitro assays, pharmacology, and toxicology study design. This interaction is an ideal opportunity to identify design problems before embarking on these experiments. When you have a complete or partial preclinical data set, a pre-IND meeting can be requested. During this meeting, you can discuss your results, strategy, and approaches to obtain FDA support. These types of discussions help to identify and avoid unnecessary studies. The FDA will provide written constructive feedback. Naturally, incorporating this feedback is crucial and avoids the possibility of a potential clinical hold in the future.
The IND application includes the nonclinical data, manufacturing conditions, and clinical protocols. All of this information is available on the FDA website. Ensure to have checked any updated regulatory guidelines, forms, and flow of the process before assembling the IND package.
Regenerative medicine advanced therapy (RMAT) designation is for drugs that are intended to treat or cure severe and life-threatening diseases. If you fulfill this designation, then your program has eligibility for increased early interaction with the FDA, priority review, and accelerated approval. The timeline of interactions with the FDA and accelerated pathways are represented in Figure 3.
The FDA also offers multiple expedited review programs to promote rare disease research. You can apply for all of them based upon the phase of product development. For example, you can apply for breakthrough designation during IND filing, and priority review during new drug application (NDA) or BLA filing. All these programs have multiple financial advantages. Orphan drug designation programs provide seven years of market exclusivity, tax credits, fi ling fee waiver, and additional assistance from the Office of Orphan Product Development. Similarly, rare pediatric disease priority review is a special program for rare diseases in the pediatric population.
The road to approval is challenging but risks can be mitigated through careful planning. Whichever strategy to product approval is ultimately chosen, keeping the end in mind will often provide the best outcome.
References
- Q3 2019 Data Report - Alliance for Regenerative Medicine [Internet]. [cited 2020 Mar 1]. Available from: https://alliancerm.org/publication/q3-2019-data-report/
- Gene Therapy Clinical Trials Worldwide [Internet]. [cited 2020 May 7]. Available from: http://www.abedia.com/wiley/vectors.php
- Li C, Samulski RJ. Engineering adeno-associated virus vectors for gene therapy. Nature Reviews Genetics. 2020 Apr;21(4):255–72.
- Limberis MP, Tretiakova A, Nambiar K, Wong G, Racine T, Crosariol M, et al. Adenoassociated virus serotype 9-expressed ZMapp in mice confers protection against systemic and airway-acquired Ebola virus infection. J Infect Dis. 2016 Dec 15;214(12):1975–9.
- Lisowski L, Dane AP, Chu K, Zhang Y, Cunningham SC, Wilson EM, et al. Selection and evaluation of clinically relevant AAV variants in a xenograft liver model. Nature. 2014 Feb 20;506(7488):382–6.
- Zincarelli C, Soltys S, Rengo G, Rabinowitz JE. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther. 2008 Jun;16(6):1073–80.
- Vandenberghe LH, Xiao R, Lock M, Lin J, Korn M, Wilson JM. Efficient serotype-dependent release of functional vector into the culture medium during adeno-associated virus manufacturing. Hum Gene Ther. 2010 Oct;21(10):1251–7.
- https://pubmed.ncbi.nlm.nih.gov/30833387/
- Zhang P, Sun B, Osada T, Rodriguiz R, Yang XY, Luo X, et al. Immunodominant liver-specific expression suppresses transgene-directed immune responses in murine Pompe disease. Hum Gene Ther. 2012 May;23(5):460–72.
- Gupta V, Cadieux CL, McMenamin D, Medina-Jaszek CA, Arif M, Ahonkhai O, et al. Adeno-associated virus-mediated expression of human butyrylcholinesterase to treat organophosphate poisoning. PLOS ONE. 2019 Nov 25;14(11): e0225188.
- https://www.pnas.org/content/115/36/E8547/tab-article-info
- Creating A Roadmap for The Development and Manufacture of Gene Therapies [Internet]. [cited 2020 May 7]. Available from: https://www.cellandgene.com/doc/creating-aroadmap-for-the-development-and-manufacture-of-gene-therapies-0001
- AAV VECTOR MANUFACTURING - Challenges & Opportunities in the Manufacturing of AAV Vectors Used in the Delivery of Gene Therapy Treatments [Internet]. Drug Development and Delivery. 2017 [cited 2020 May 8]. Available from: https://drug-dev.com/aav-vectormanufacturing-challenges-opportunities-in-the-manufacturing-of-aav-vectors-used-inthe-delivery-of-gene-therapy-treatments/
- REGENXBIO and Biogen Enter Exclusive License Agreement for the Development of Gene Therapy Treatments for Rare Genetic Vision Disorders [Internet]. REGENXBIO Inc. [cited 2020 May 8].
- Available from: http://ir.regenxbio.com/news-releases/news-release-details/regenxbioand-biogen-enter-exclusive-license-agreement