Safety First: Perspective on Patient-Centered Development of AAV Gene Therapy Products
Authors: Barry J. Byrne
Journal: Molecular Therapy (2018)

Recent publications in Molecular Therapy1Hordeaux J. Wang Q. Katz N. Buza E.L. Bell P. Wilson J.M. The neurotropic properties of AAV-PHP.B are limited to C57BL/6J mice.Mol Ther. 2018; 26 (this issue): 664-668Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar and Human Gene Therapy2Hinderer C. Katz N. Buza E.L. Dyer C. Goode T. Bell P. Richman L.K. Wilson J.M. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an AAV vector expressing human SMN.Hum. Gene Ther. 2018; (Published online February 12, 2018)https://doi.org/10.1089/hum.2018.015Crossref Scopus (379) Google Scholar identify acute toxicity of adeno-associated virus (AAV)-mediated survival of motor neuron (SMN) or GFP expression, which warrants careful consideration to better understand the observation as well as the need to perform well-controlled studies to address this finding. However, these findings should be viewed in the context of an initial observation in a basic proof-of-concept study and not in relationship to established practices for rational and patient-centered drug development, which follow guidance from the International Committee on Harmonization (ICH) for Good Clinical Practice (GCP).3FDA (1996). Guidance for industry E6 good clinical practice: consolidated guidance. https://www.fda.gov/downloads/drugs/guidances/ucm073122.pdf.Google Scholar The purpose of this commentary is to address the basis by which gene therapy medicinal products are evaluated in non-clinical studies to best inform us of the risk-benefit profile of a given product under development for a specific indication. The findings of the two publications can be summarized as preliminary studies to develop an approach to spinal muscular atrophy (SMA) using variants of AAV9. The first study tested these vectors in piglets and non-human primates (NHPs) with a finding of acute toxicity in one NHP and neural inflammation in the piglets. The second study showed a single animal with the AAV9 variant, PHP.B, which developed an acute coagulopathy. Importantly, the severe findings in a limited number of animals in these studies should not be confused with low-grade laboratory findings in clinical studies that are self-limited. Since these studies are related to variants of AAV9 vectors given systemically, the focus of this commentary will be limited to active clinical programs using AAV serotype 9, both by systemic and regional delivery, since the one conclusion of Hinderer et al.2Hinderer C. Katz N. Buza E.L. Dyer C. Goode T. Bell P. Richman L.K. Wilson J.M. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an AAV vector expressing human SMN.Hum. Gene Ther. 2018; (Published online February 12, 2018)https://doi.org/10.1089/hum.2018.015Crossref Scopus (379) Google Scholar is that, “The present results and those of another recent study utilizing a different AAV9 variant and transgene indicate that systemic and sensory neuron toxicity may be general properties of intravenous delivery of AAV vectors at high doses irrespective of the capsid serotype or transgene.”1Hordeaux J. Wang Q. Katz N. Buza E.L. Bell P. Wilson J.M. The neurotropic properties of AAV-PHP.B are limited to C57BL/6J mice.Mol Ther. 2018; 26 (this issue): 664-668Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 2Hinderer C. Katz N. Buza E.L. Dyer C. Goode T. Bell P. Richman L.K. Wilson J.M. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an AAV vector expressing human SMN.Hum. Gene Ther. 2018; (Published online February 12, 2018)https://doi.org/10.1089/hum.2018.015Crossref Scopus (379) Google Scholar Platform technology studies are important to understanding basic behavior of AAV-vectors in vivo. However, in order to develop therapies aimed at treating human disease, it is imperative that investigators follow the guidance agreed to by Federal Drug Administration (FDA) and European Medicines Agency (EMA) which oversee human clinical research conducted under the principles of GCP. The non-clinical and clinical data supporting nine active clinical studies should also be considered in evaluating the clinical application of AAV9 vectors. GCP guidance is based on the ethical principles established in the Declaration of Helsinki in which the rights, safety, and well-being of trial subjects are the most important and overriding considerations in the design and conduct of research involving human subjects. Therefore, any anticipated risk should be established in light of the potential benefit for the subject. This principle is further emphasized in research involving children, the greatest proportion of those with severe genetic conditions amenable to gene therapy, where the prospect of direct benefit is required to conduct a pediatric study. Because of these principles, all available non-clinical and clinical data for an investigational product should directly support the proposed clinical use. The guidelines provide specific recommendations on how the investigator, institutional review board (IRB), independent ethics committee (IEC) and biosafety committee is informed of the non-clinical findings by way of the Investigators Brochure (IB). The IB provides information on the characteristics of the investigational product and the non-clinical studies which support the clinical use. Therefore, a comparison of the cumulative findings from IBs which support active AAV9 studies to the current non-clinical studies by Hordeux et al.1Hordeaux J. Wang Q. Katz N. Buza E.L. Bell P. Wilson J.M. The neurotropic properties of AAV-PHP.B are limited to C57BL/6J mice.Mol Ther. 2018; 26 (this issue): 664-668Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar and Hinderer et al.2Hinderer C. Katz N. Buza E.L. Dyer C. Goode T. Bell P. Richman L.K. Wilson J.M. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an AAV vector expressing human SMN.Hum. Gene Ther. 2018; (Published online February 12, 2018)https://doi.org/10.1089/hum.2018.015Crossref Scopus (379) Google Scholar is warranted. The findings in the two publications are related to “an attempt to develop PHP.B for human gene therapy”1Hordeaux J. Wang Q. Katz N. Buza E.L. Bell P. Wilson J.M. The neurotropic properties of AAV-PHP.B are limited to C57BL/6J mice.Mol Ther. 2018; 26 (this issue): 664-668Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar and therefore an evaluation of efficacy and toxicity in nonhuman primates (NHPs) was done using AAV9 and two variants4Deverman B.E. Pravdo P.L. Simpson B.P. Kumar S.R. Chan K.Y. Banerjee A. Wu W.L. Yang B. Huber N. Pasca S.P. Gradinaru V. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.Nat. Biotechnol. 2016; 34: 204-209Crossref PubMed Scopus (528) Google Scholar which were anticipated to enhance CNS gene transfer following systemic delivery of the vector. The findings of the paper in Human Gene Therapy are the subject of a commentary by Flotte and Buning5Flotte T.R. Buning H. Severe toxicity in nonhuman primates and piglets with systemic high-dose administration of adeno-associated virus serotype 9-like vectors: putting patients first.Hum. Gene Ther. 2018; (Published online February 7, 2018)https://doi.org/10.1089/hum.2018.021Crossref Scopus (16) Google Scholar and, in reference to the study in Molecular Therapy, there are two key observations. First, a well-designed set of experiments confirmed the interesting finding that the enhanced activity of the PHP.B variant is limited to the mouse strain in which it was developed. The second finding is significantly limited by the use of a single animal per group and, using GFP expression as the marker of gene transfer at two dose levels, modeled on the prior experience in humans with AAV-SMN.6Mendell J.R. Al-Zaidy S. Shell R. Arnold W.D. Rodino-Klapac L.R. Prior T.W. Lowes L. Alfano L. Berry K. Church K. et al.Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy.N. Engl. J. Med. 2017; 377: 1713-1722Crossref PubMed Scopus (1182) Google Scholar In the initial dose group, GFP expression was detected at 21 days after delivery and therefore it is not surprising that gene expression would have begun even sooner in the higher dose group, since plus/minus strand annealing allows for rapid transgene expression when AAV dose is at the level of dose saturation. Specific assays for anti-capsid or anti-GFP immune response are not presented. The rapidity of the response may be related to immune activation of compliment or oxidative damage in hepatocytes due to impurities in the vector preparation. Immuno-toxicity can in fact be managed by immune modulation which would allow for incremental dosing.7Corti M. Cleaver B. Clément N. Conlon T.J. Faris K.J. Wang G. Benson J. Tarantal A.F. Fuller D. Herzog R.W. Byrne B.J. Evaluation of Readministration of a Recombinant Adeno-Associated Virus Vector Expressing Acid Alpha-Glucosidase in Pompe Disease: Preclinical to Clinical Planning.Hum. Gene Ther. Clin. Dev. 2015; 26: 185-193Crossref PubMed Scopus (56) Google Scholar, 8Corti M. Liberati C. Smith B.K. Lawson L.A. Tuna I.S. Conlon T.J. Coleman K.E. Islam S. Herzog R.W. Fuller D.D. et al.Safety of Intradiaphragmatic Delivery of Adeno-Associated Virus-Mediated Alpha-Glucosidase (rAAV1-CMV-hGAA) Gene Therapy in Children Affected by Pompe Disease.Hum. Gene Ther. Clin. Dev. 2017; 28: 208-218Crossref PubMed Scopus (63) Google Scholar An additional challenge is that the vector production and quantification was accomplished by a different method than established for other clinical programs which followed Good Laboratory Practice (GLP) guidance, especially related to a qualified assay for characterization of vector quantity. Therefore, reliance on the results of these studies should be limited to understanding the means by which an acute response leading to coagulopathy can be explained in NHPs and the unique finding of strain specific murine receptor for AAV PHP.B. In contrast, nine active clinical programs are supported by non-clinical data showing product safety in excess of the clinical dose used in those clinical studies, often in multiple species. The ascending dose study design used in most early phase studies is required by regulators in order to establish the most efficacious dose in the absence of dose limiting toxicity. Table 1 lists the study number in ClinicalTrials.gov, indication, and supporting non-clinical data. Among these studies, the recent publication in NEJM by Mendell et al.6Mendell J.R. Al-Zaidy S. Shell R. Arnold W.D. Rodino-Klapac L.R. Prior T.W. Lowes L. Alfano L. Berry K. Church K. et al.Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy.N. Engl. J. Med. 2017; 377: 1713-1722Crossref PubMed Scopus (1182) Google Scholar confirms not only the safety of AVXS-101 (AAV9-SMN) therapy but also a striking clinical benefit in a vulnerable patient population under six months of age. The findings of this study and the others underway confirm the important underlying principle of GCP: that risk-benefit must be in favor of a clinically meaningful benefit to participants in the study.Table 1Listing of Active AAV9 Studies in Clinicaltrials.govStudy NumberTitleAAV9 VectorIndicationNHPs/CanineRodentsNCT02240407Re-administration of Intramuscular AAV9 in Patients with Late-Onset Pompe DiseaseAAV-GAAPompe Disease28280NCT02725580Batten CLN6 Gene TherapyAAV-CLN6Batten Diseasen/an/aNCT03381729Study of Intrathecal Administration of AVXS-101 for Spinal Muscular AtrophyAVXS-101AAV-SMNSMAn/an/aNCT02362438Intrathecal Administration of scAAV9/JeT-GAN for the Treatment of Giant Axonal NeuropathyscAAV9/JeT-GANGAN14180NCT03368742Microdystrophin Gene Transfer Study in Adolescents and Children with DMDSGT-001DMD16/21500NCT03362502A Study to Evaluate the Safety and Tolerability of PF-06939926 Gene Therapy in Duchenne Muscular DystrophyPF-06939926DMDn/an/aNCT03306277Gene Replacement Therapy Clinical Trial for Patients with Spinal Muscular Atrophy Type 1AVXS-001SMA4403NCT02716246Phase I/II Gene Transfer Clinical Trial of scAAV9.U1a.hSGSH for Mucopolysaccharidosis (MPS) IIIAscAAV9.U1a.hSGSHMPSIII An/an/aNCT03315182Phase I/II Gene Transfer Clinical Trial of AAV9.CMV.hNAGLU for Mucopolysaccharidosis (MPS) IIIBrAAV9.CMV.hNAGLUMPSIII B8n/a Open table in a new tab The regulatory framework which requires the rigorous review of proof-of-concept data and product specific toxicology and biodistributoin studies is the basis for which clinical research with AAV vectors can be accomplished by emphasizing safety as the first priority.9Lima BSV M.A. Toxicology and Biodistribution: The Clinical Value of Animal Biodistribution Stuides.Mol. Ther. Methods Clin. Dev. 2018; (Published online January 31, 2018)https://doi.org/10.1016/j.omtm.2018.01.003Abstract Full Text Full Text PDF Scopus (22) Google Scholar It is important for all those in the field of gene therapy to be vigilant regarding findings related to product safety. An additional tenant of GCP guidance is that the medical care provided to subjects who volunteer for clinical research studies be provided by a qualified physician. In my own experience of providing medical supervision for over 50 individuals enrolled in a variety of clinical studies using AAV-mediated gene transfer, each program requires a detailed knowledge of the supporting literature, the IB, and the product characteristics (Chemistry, Manufacturing, and Control [CMC]), all of which establish the dose, potency, and safety by the proposed route of administration. In the current era of clinical implementation of AAV-mediated gene transfer we must rely on all those in the field to conduct the most informative basic and clinical investigation with the common goal of bringing much needed innovative medical therapy to patients who have a high burden of disease. By following a patient-focused and rational drug development path, the gene therapy community will be successful in meeting the long anticipated expectation of targeted precision medicine using gene therapy. B.J.B. is an inventor of intellectual property owned by the Johns Hopkins University and the University of Florida related to this manuscript. He is a member of the Pfizer Rare Disease Therapeutic Advisory Board and co-founder of Lacerta Therapeutics. The author is also an unpaid member of two AAV9 DSMBs and the principal investigator of Ignite DMD (NCT03368742) for which there are no competing interests. The Neurotropic Properties of AAV-PHP.B Are Limited to C57BL/6J MiceHordeaux et al.Molecular TherapyFebruary 1, 2018In BriefImproved delivery of adeno-associated virus (AAV) vectors to the CNS will greatly enhance their clinical utility. Selection of AAV9 variants in a mouse model led to the isolation of a capsid called PHP.B, which resulted in remarkable transduction of the CNS following intravenous infusion. However, we now show here that this enhanced CNS tropism is restricted to the model in which it was selected, i.e., a Cre transgenic mouse in a C57BL/6J background, and was not found in nonhuman primates or the other commonly used mouse strain BALB/cJ. Full-Text PDF Open Archive