David W. Souza

Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis

The gene associated with cystic fibrosis (CF) encodes a membrane-associated, N-linked glycoprotein called CFTR. Mutations were introduced into CFTR at residues known to be altered in CF chromosomes and in residues believed to play a role in its function. Examination of the various mutant proteins in COS-7 cells indicated that mature, fully glycosylated CFTR was absent from cells containing delta F508, delta 1507, K464M, F508R, and S5491 cDNA plasmids. Instead, an incompletely glycosylated version of the protein was detected. We propose that the mutant versions of CFTR are recognized as abnormal and remain incompletely processed in the endoplasmic reticulum where they are subsequently degraded. Since mutations with this phenotype represent at least 70% of known CF chromosomes, we argue that the molecular basis of most cystic fibrosis is the absence of mature CFTR at the correct cellular location.

Preexisting Immunity and Low Expression in Primates Highlight Translational Challenges for Liver-directed AAV8-mediated Gene Therapy

Liver-directed gene therapy with adeno-associated virus (AAV) vectors effectively treats mouse models of lysosomal storage diseases (LSDs). We asked whether these results were likely to translate to patients. To understand to what extent preexisting anti-AAV8 antibodies could impede AAV8-mediated liver transduction in primates, commonly preexposed to AAV, we quantified the effects of preexisting antibodies on liver transduction and subsequent transgene expression in mouse and nonhuman primate (NHP) models. Using the highest viral dose previously reported in a clinical trial, passive transfer of NHP sera containing relatively low anti-AAV8 titers into mice blocked liver transduction, which could be partially overcome by increasing vector dose tenfold. Based on this and a survey of anti-AAV8 titers in 112 humans, we predict that high-dose systemic gene therapy would successfully transduce liver in >50% of human patients. However, although high-dose AAV8 administration to mice and monkeys with equivalent anti-AAV8 titers led to comparable liver vector copy numbers, the resulting transgene expression in primates was ~1.5-logs lower than mice. This suggests vector fate differs in these species and that strategies focused solely on overcoming preexisting vector-specific antibodies may be insufficient to achieve clinically meaningful expression levels of LSD genes using a liver-directed gene therapy approach in patients.

92. Evaluation of Producer Cell Line Platform for Production of Oversized AAV-FVIII Vectors

Recombinant adeno-associated virus (rAAV) vectors are being evaluated as gene delivery vehicles in several clinical trials. The 4.7 kb wild-type (WT) size genome of AAV presents a challenge for incorporating larger transgenes with incomplete vector genome (vg) packaging being a frequent outcome. To test the feasibility of producing oversized rAAV production using the producer cell line (PCL) method, we generated slightly oversized rAAV vectors (harboring 5.1 or 5.4 kb sized vgs) containing a liver-restricted promoter (mTTR) and a codon-optimized cDNA encoding human B-domain deleted FVIII (FVIIIco). Genomes were packaged into the AAVrh8R serotype vector using the PCL process and compared to matched vectors generated via the triple transfection (TXN) method. Vectors were then characterized for production yields, integrity of packaged genomes and homogeneity. The data showed that the PCL platform was able to produce oversized AAV vectors at levels that were 10- to 100-fold higher than the TXN process with yields greater than 100,000 vg/cell. The PCLs were stable with consistent production maintained up to 60 passages. Southern and dot blot analyses of the packaged genomes demonstrated encapsidation of genomes larger than 4.7 kb in the PCL generated vector while the majority of genomes packaged via the TXN method were 4.7 kb in size. Furthermore, the PCL process generated more vector DNA-containing particles and less packaging of non-vector DNA. Testing the PCL generated vectors in the hemophilia A knock-out (KO) mouse model showed a 2-fold higher plasma FVIII activity (Coatest) and vg copies in the livers than obtained with the vectors made by the TXN process. In summary, the PCL production process generated higher yields of oversized rAAV/FVIIIco vectors as well as higher quality vectors than the TXN method. Hence, the PCL platform may be used for producing greater quality oversized rAAV vectors at levels that can meet the needs for clinical studies.

245. AAV2/8-Mediated Efficacy and Immune Tolerance in a Pompe Disease Mouse Model

Pompe disease is an inherited metabolic disorder caused by a deficiency of the lysosomal enzyme acid -glucosidase (GAA). The disease is characterized by accumulation of glycogen in various tissues, including the central and peripheral nervous systems, but the pathology most dramatically affects skeletal and cardiac muscles. Severely affected patients present within the first year of life with hypotonia and cardiomyopathy, resulting in death from cardiopulmonary failure. To assess the utility of gene therapy for Pompe disease, we generated recombinant AAV8-based serotype vectors encoding for GAA under the transcriptional control of a liver-restricted promoter (DC190). Systemic administration of 5X1011 drp of AAV8/DC190-GAA into Pompe mice generated high levels (25-30 g/ml) of GAA in the serum that persisted for 6 months post-administration. GAA activity was also detected in several muscle groups isolated from the AAV8-treated mice, indicating that GAA was efficiently taken up into these tissues from circulation. Glycogen storage was completely corrected in the heart and diaphragm and reduced by 50% in the skeletal muscles as early as 4 weeks post-treatment. All tissues were completely cleared of glycogen storage by 10 weeks, and remained at baseline at the 6-month time point. Histomorphometric analysis of PAS-stained tissues confirmed that glycogen deposits were effectively cleared from the affected tissues. Histopathology observed in the dorsal root ganglia of Pompe mice was also normalized suggesting that treatment may impact the peripheral nerve disease. Consistent with previous studies using the DC190 promoter, no antibodies to the human GAA were detected in the sera of AAV8/DC190-GAA-treated mice. Furthermore, no antibodies were generated following two subsequent challenges with recombinant GAA emulsified in Complete Freunds adjuvant, suggesting the AAV8/DC190-GAA-treated mice were immune tolerized. Immune responses were also assessed at the cellular level by assaying lymphocyte proliferation and IFN secretion using cells harvested from the spleens of the treated mice. Cells from the AAV8/DC190-GAA-treated mice showed an attenuated response to antigenic stimulation compared to controls. These data support the continued evaluation of AAV8-mediated gene therapy as an approach to treat the systemic manifestations of Pompe disease.