Considerations for Systemic Use of Gene Therapy
Authors: Barry J. Byrne, Manuela Corti, Francesco Muntoni
Journal: Molecular Therapy (2021)

Many early-onset genetic diseases are rapidly progressive fatal conditions for which there has been limited or no therapeutic option. However, systemic gene therapy using adeno-associated viral (AAV) vectors has emerged as a viable therapeutic strategy owing to the long duration of effect of an AAV vector targeted to a terminally differentiated cell. The goal of systemic delivery is to reach body-wide cellular targets that are impacted by the underlying mutation and restore gene function. Sustained effects are more challenging in cell populations where cell growth is ongoing (such as in the musculature) since the AAV genomes remain episomal and are subject to dilution with nuclear division. In this paper, we consider the implications of systemic exposure to AAV vectors and the need for caution in implementation of these powerful tools to combat genetic disease using gene augmentation therapy and gene editing approaches that rely on AAV vector technology. Improved understanding of the genetic basis and molecular pathogenesis for the majority of the severe and devastating inherited conditions is leading to very rapid progress in the development of AAV gene therapy for several previously untreatable conditions. Advances are also facilitated by the characterization of the different cellular tropisms of myriad naturally occurring or experimentally evolved AAV serotypes. By combining capsid tropism and promoter engineering with a deep understanding of the underlying biology of the disease being treated, first generation systemic AAV gene therapies have reached advanced stages of development or have received regulatory approval.1Hoy S.M. Onasemnogene Abeparvovec: First Global Approval.Drugs. 2019; 79: 1255-1262Crossref PubMed Scopus (114) Google Scholar When considering any new therapeutic approach to a systemic disease, a primary consideration that requires careful review is the ratio of risk to the potential for direct benefit. In fact, strict regulatory requirements (subpart D; Code of Federal Regulations [CFR] 46.405) for the prospect of direct benefit is a common aspect of any pediatric study population. Recent findings from several pediatric studies have led to a reexamination of the risk:benefit ratio, especially related to immunotoxicity following systemic use of AAV vectors. Careful consideration should be given to the immunotoxicity associated with systemic delivery of gene therapy vectors in the context of older patients with more advanced and chronic forms of disease (also with greater total capsid burden).2Kirschner J. Butoianu N. Goemans N. Haberlova J. Kostera-Pruszczyk A. Mercuri E. van der Pol W.L. Quijano-Roy S. Sejersen T. Tizzano E.F. et al.European ad-hoc consensus statement on gene replacement therapy for spinal muscular atrophy.Eur. J. Paediatr. Neurol. 2020; 28: 38-43Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar The greatest experience to date has been with AAV9 vectors, especially for spinal muscular atrophy (SMA), with over 400 doses administered; however, these findings are not limited to AAV9. Numerous reports of serious adverse events (including three recent deaths in a study for myotubular myopathy) have a common theme of immunotoxicity in the liver, sensory ganglia,3Mueller C. Berry J.D. McKenna-Yasek D.M. Gernoux G. Owegi M.A. Pothier L.M. Douthwright C.L. Gelevski D. Luppino S.D. Blackwood M. et al.SOD1 Suppression with Adeno-Associated Virus and MicroRNA in Familial ALS.N. Engl. J. Med. 2020; 383: 151-158Crossref PubMed Scopus (36) Google Scholar and heart and acute kidney injury following complement activation related to thrombotic microangiopathy.4Chand D.H. Zaidman C. Arya K. Millner R. Farrar M.A. Mackie F.E. Goedeker N.L. Dharnidharka V.R. Dandamudi R. Reyna S.P. Thrombotic Microangiopathy Following Onasemnogene Abeparvovec for Spinal Muscular Atrophy: A Case Series.J. Pediatr. 2020; (Published online November 28, 2020)https://doi.org/10.1016/j.jpeds.2020.11.054Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Added perspective on the importance of adverse immune responses can be drawn from the experience of solid organ transplantation developed approximately 60 years ago. Organ transplantation—like gene therapy—has had an enormous impact on medical practice, and both therapies are a one-way path in which the treatment cannot be reversed. Non-clinical studies in transplant and some early clinical experience recognized the consequence of immune-mediated graft failure. Successful solid organ transplant required a more thorough understanding of immunology and ways to mitigate acute rejection. After nearly a decade of failures, multi-drug regimens were developed to promote graft survival. At the same time, improved donor matching led to further improvements, and the analogy to gene therapy is evident in the use of anti-AAV immune assays as a qualification for gene therapy. Corticosteroid therapy was used in most of the early transplant efforts, yet it was quickly learned that prolonged use of high-dose steroids also had negative consequences, such as susceptibility to infection and systemic side effects. Attempts to increase the potency and specificity of gene therapy products is undergoing rapid development to improve efficiency and hopefully lessen the immune response profile. Nonetheless, the adaptive humoral immune response to viral capsid proteins can produce an infinite amount of anti-capsid antibody. The total capsid exposure at dosing is the product of vector genome (vg) dose per kg, patient weight, vg concentration, and total protein concentration of the vector product (a surrogate for the empty:full ratio of AAV vectors). The total capsid protein load in a typical systemic dose is 50–100 times that of a typical childhood immunization, so it is not surprising that a strong reaction is elicited. Certainly, higher total capsid load is more likely to result in immunotoxicity where the capsid exposure and biodistribution is the greatest (e.g., the liver, sensory ganglia, and heart). These considerations come into play after the primary exposure, where antibody concentration increases rapidly (4–5 log orders) within the first few days after dosing. Characterization of antibody and vector pharmacokinetics in the blood had not been thoroughly examined due to practical considerations of repeated mouse bleeds or need for general anesthesia in non-human primate (NHP) studies. Only through recent studies in human subjects has it become clear that events within the first week after dosing strongly influence the onset of adverse events related to immunotoxicity. Lastly, we need to consider more carefully the differences in non-clinical models in relation to the human subject population. Body weight normalization does not take into account the issue of total exposure (e.g., a 3-kg NHP is not representative of a 40–60 kg child or young adult). Also, there are vast differences in the organ:body weight ratio in various species as well as at different developmental stages (the human infant brain is 10% of body weight and only 2% of body weight in adults). An additional issue that requires careful consideration relates to the consequences that different gene mutations have on the ability of the patients to produce the protein. For example, patients carrying missense mutations can produce low or even normal levels of mutant (non-functional) protein, whereas patients carrying null alleles produce no protein. The lack of any cross-reactive immunological material (CRIM) could lead to the development of an anti-transgene response, adding another layer of complexity to the immunological considerations with the risk of acute or chronic rejection. These factors lead us to consider the need for immune management and protection from safety events soon after dosing. Additionally, duration of effect is influenced by the non-integrating nature of AAV vectors. Terminally differentiated cells, such as neurons and cardiomyocytes, provide the highest confidence of AAV and transgene durability, although some consideration for a low grade of cardiomyocyte renewal in the first decade of life also highlights the need to consider age at intervention.5Bergmann O. Bhardwaj R.D. Bernard S. Zdunek S. Barnabé-Heider F. Walsh S. Zupicich J. Alkass K. Buchholz B.A. Druid H. et al.Evidence for cardiomyocyte renewal in humans.Science. 2009; 324: 98-102Crossref PubMed Scopus (2058) Google Scholar The concept is more acute when considering skeletal muscle; while the number of skeletal muscle cells is determined at birth, the massive increase in width and length that occurs in these multinucleated cells between the neonatal period and adolescence leads to the inevitable dilution of the transgene.6Yin H. Price F. Rudnicki M.A. Satellite cells and the muscle stem cell niche.Physiol. Rev. 2013; 93: 23-67Crossref PubMed Scopus (993) Google Scholar Interestingly, the precise figure on the nuclear accretion between newborn and adult muscle has been studied well in several animal species but is not well understood in humans. These considerations raise the question of whether some skeletal muscle conditions should be treated with AAV shortly after birth (for example, following a newborn screening program), when the target tissue is in optimal histological condition, or to wait a few years or longer, when there is less well-preserved skeletal muscle but a larger number of nuclei, so as to presumably minimize the dilution effect. Providing an opportunity for re-administration of AAV vectors is the most rational approach to achieve early correction and preserve the option for re-exposure over the course of the lifespan. In summary, an enormous opportunity exists to change medical practice with transformative treatments using systemic gene therapy. At the same time, there has to be consideration of the need for targeted immunotherapy to lessen the innate and early adaptive immune responses following systemic delivery. We propose that well-designed and targeted immunotherapy will enhance both safety and efficacy in systemic gene therapy and allow for broad access to gene therapy for a wide variety of conditions in all ages and disease types.