The following proposal builds and expands on the results obtained from the preclinical and clinical trial conducted on the previous grant cycles. An initial bridging clinical study of rAAV1-AAT via isolated limb perfusion (ILP) delivery is planned in the first year of the application. This small clinical study will directly compare the safety and gene transfer efficiency in matched AAT-deficient volunteers receiving the previously used dosages level of rAAV1-AAT, but now produced by the HSV-recombinant method. If this comparison is favorable, future clinical trials will use the vector produced by the HSV method and administered by the ILP technique. We also propose to expand the armamentarium of rAAV muscle directed gene therapy for AAT by performing preclinical testing and characterization of rAAV9. This serotype has demonstrated superiority to rAAV1 in a number of studies. In order to exploit this advantage in our current clinical program, preclinical toxicology studies will continue to be performed in a GLP compliant manner. We propose to perform preclinical toxicology and biodistribution studies in C57BL6 mice and baboons. The mouse studies will utilize the same rAAV9-AAT vector to be used for the future clinical trials, taking advantage of the fact that C57BL6 mice are naturally tolerant to human AAT. These studies will seek to compare the efficiency of ILP delivery of rAAV1 and rAAV9 as a means to get broader transduction of skeletal muscle as compared with direct IM delivery of rAAV1-AAT, which is the currently active clinical approach. This work will also establish the safety and biodistribution parameters of rAAV9-AAT. We then propose to study the delivery of rHSV-produced rAAV9-AAT via isolated limb perfusion to a cohort of chimpanzees. This model will allow us to characterize immune responses by measuring ELISPOT responses to AAV9 capsid proteins and to AAT transgene product as well as antibody formation against rAAV9 capsid. Ultimately the success of a gene therapy strategy depends on the ability of the vector to deliver the therapeutic gene to the target cell population in a safe and efficient manner. Immune responses to the gene therapy vector can eliminate the vector and transfected cells which in turn decreases the intensity and duration of protein expression. In our last aim we propose to study the effects of transient immune suppression on the duration and intensity of AAT production by rAAV9 ILP delivery in chimpanzees by depleting select lymphocyte populations in 4 different cohorts. CD8 T cell depletion will be used to determine the limiting effect cell mediated responses will have on transgene expression and duration. Likewise the role of humoral and T helper cells will be studied by the depletion of B cells and CD4 respectively. The fourth cohort of this aim will explore the possibility of blocking antigen presentation by transient blockage of co-stimulatory signals needed for successful T and B cell activation which may help lead to long term survival of transfected cells or perhaps even tolerance to rAAV9. The ultimate goal of proposal will be to lead into a future clinical trial of ILP delivery of rAAV9-AAT produced by the HSV-helper system, thus allowing for an increase in both the """"""""potency"""""""" of the vector (anticipating greater AAT expression per vector genome from the rAAV9 ILP approach) and the total dose deliverable (anticipation greater scalability of the rAAV9-based material and the improvement of delivery by ILP vs. IM injection). However, until the results of the studies proposed in aims 1 and 2 are completed, it is premature to propose a specific rAAV9-ILP trial. In addition, the issues to be investigated in aims 3 and 4, i.e., the immune response questions, will also be essential in developing the appropriate strategy for later clinical trials.

Public Health Relevance

Individuals with a deficiency of the Alpha 1-antitrypsin (AAT) protein are at risk for developing emphysema and liver damage. AAT deficiency is a genetic disorder in which individuals have inadequate levels of the AAT protein. AAT protects the lungs from white blood cell enzymes that can damage air sacs within the lungs, potentially leading to emphysema. Experimental gene transfer procedures, in which normal copies of genes are inserted into cells, are being developed to treat many genetic diseases, including AAT deficiency. In this proposal, a modified virus, adeno-associated virus (AAV), has been genetically engineered to contain a normal copy of the AAT gene. When AAV is combined with the AAT gene, the resulting agent, rAAV-CB-hAAT, is able to carry normal copies of the AAT gene into muscle cells to produce additional AAT. The purpose of this proposal is to evaluate the safety of injecting rAAV-CB-hAAT into individuals with AAT deficiency.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Gene and Drug Delivery Systems Study Section (GDD)
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Croxton, Thomas
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University of Massachusetts Medical School Worcester
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Mueller, Christian; Gernoux, Gwladys; Gruntman, Alisha M et al. (2017) 5 Year Expression and Neutrophil Defect Repair after Gene Therapy in Alpha-1 Antitrypsin Deficiency. Mol Ther 25:1387-1394
Gruntman, Alisha M; Su, Lin; Su, Qin et al. (2015) Stability and compatibility of recombinant adeno-associated virus under conditions commonly encountered in human gene therapy trials. Hum Gene Ther Methods 26:71-6
Gruntman, Alisha M; Flotte, Terence R (2015) Progress with Recombinant Adeno-Associated Virus Vectors for Gene Therapy of Alpha-1 Antitrypsin Deficiency. Hum Gene Ther Methods 26:77-81
Loring, Heather S; Flotte, Terence R (2015) Current status of gene therapy for ?-1 antitrypsin deficiency. Expert Opin Biol Ther 15:329-36
Gruntman, Alisha M; Flotte, Terence R (2015) Delivery of Adeno-Associated Virus Gene Therapy by Intravascular Limb Infusion Methods. Hum Gene Ther Clin Dev 26:159-64
Flotte, Terence R; Mueller, Christian (2014) What is suppression of anti-adeno-associated virus capsid T-cells achieving? Hum Gene Ther 25:178-9
Gruntman, Alisha M; Bish, Lawrence T; Mueller, Christian et al. (2013) Gene transfer in skeletal and cardiac muscle using recombinant adeno-associated virus. Curr Protoc Microbiol Chapter 14:Unit 14D.3
Mueller, Christian; Chulay, Jeffrey D; Trapnell, Bruce C et al. (2013) Human Treg responses allow sustained recombinant adeno-associated virus-mediated transgene expression. J Clin Invest 123:5310-8
Mueller, Christian; Tang, Qiushi; Gruntman, Alisha et al. (2012) Sustained miRNA-mediated knockdown of mutant AAT with simultaneous augmentation of wild-type AAT has minimal effect on global liver miRNA profiles. Mol Ther 20:590-600
Flotte, Terence R; Trapnell, Bruce C; Humphries, Margaret et al. (2011) Phase 2 clinical trial of a recombinant adeno-associated viral vector expressing ?1-antitrypsin: interim results. Hum Gene Ther 22:1239-47

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