Beyond Gene Replacement Strategies: Safety Considerations for Novel AAV Vector Modalities

Adeno-associated virus (AAV) vectors have been widely leveraged in gene therapy, and currently a number of products, including Luxterna® and Zolgensma®, have already been approved for gene replacement therapy using AAV vector technology.  To date, AAV vectors have been the workhorse of gene therapy, with most products engineered to express a transgene that replaces a dysfunctional or missing gene. But the full potential of AAV vectors has not yet been fully explored, as these vectors hold significantly greater potential beyond standard gene replacement.  AAV vectors also provide a number of advantages over other currently available gene therapy vectors.  First, they are generally active/efficacious after a single dose, resulting in long-term expression in the patient.  AAV vectors have also been shown to be generally very safe up to extremely high systemic or local doses.  Another key advantage of these products is the numerous different, even hybrid, viral capsids which allow targeting of the vector to different tissues or combinations of tissues.  These vectors can also be engineered to be replication incompetent, so that there is little danger of an overt or pathogenic infection.  Moreover, AAV vectors remain episomal (within the cytoplasm), and do not integrate into the host cell genome, unlike many other viral vectors.  Finally, AAV gene therapy can be delivered by different routes of administration, including IV, IM, intrathecal or even intraocular, so that  specific tissue targeting can be achieved. 

However, AAV vectors exhibit their own drawbacks and disadvantages, which have limited their broader use as gene delivery vectors.  Due to the nature of their capsid size, AAV vectors can only harbor genes up to about 4.8 Kb, thus limiting them to smaller transgenes.  Also, the AAV capsids are immunogenic in practically all mammalian species, so that they cannot be dosed repeatedly, limiting them to single dose therapy.  Moreover, most humans and animal species already harbor antibodies against many AAV serotypes, so that antibody screening is crucial prior to AAV therapy.  Another disadvantage of many AAV serotypes is that upon systemic dosing, most viral particles end up in the liver, resulting in possible liver toxicity and limiting whatever remaining vector is available for the target tissue.  Although cited above as an advantage, since AAV vectors do not integrate into the genome, they can be lost over time or “diluted out” in dividing tissues.  Therefore, they may not persist for the life of the patient, and AAV vectors cannot be re-dosed due to their immunogenicity.  Finally, most vectors developed to date utilize strong, constitutive (always on) promoters which means that transgene expression cannot be “fine-tuned” for specific tissues or toned down if less gene expression is required. 

Despite these potential limitations on the expanded use of AAV vectors, a number of novel approaches are currently being leveraged to increase AAV vector utility and to expand beyond standard gene replacement approaches.  For example, novel vector capsids are being developed to decrease immunogenicity and increase tissue specificity, thereby bypassing possible liver toxicity.  Other approaches include use of small molecule gene activators which can stimulate or inhibit gene expression to more finely tune delivery of the expressed protein, or use of “dual AAV” vector approaches to express larger transgenes or multiple transgenes.  AAV vectors can also be used to express transcription factors to further edit downstream gene expression, or to express gene editing enzymes such as CRISPR-Cas9 to specifically modify the host genome to knock out or in other disease-causing genes.  Furthermore, AAV vectors may be delivered directly into key tissue compartments such as the eye or the CNS to increase the local concentration of the vector, thereby decreasing potential systemic toxicity. 

Altogether, today’s gene therapy product Sponsors need to consider these various technical developments and combinations of approaches while still demonstrating product safety and efficacy.  Leveraging novel vector capsids, split transgenes or combination AAV products will ultimately result in more complex products that may warrant additional safety testing and considerations.  Sponsors must keep these challenges in mind and consider what impact they might have on vector safety, distribution, and efficacy.  If a nonclinical program can be specifically tailored to both the vector product and patient population, Sponsors should be able to mitigate any unanticipated safety concerns and ideally bring these novel, more efficacious and safer AAV vector products through clinical development and into the patients who need them most.