1094. A Novel Replication Competent, but Packaging Deficient Adenoviral(Ad) Vector [E1+, 100K-]hGAA for High Level hGAA Expression and Reduced Toxicity in Gene Therapy of Glycogen Storage Disease II (GSD-II)
Authors: Fang Xu, Zachary Hartman, Delila Serra, Andrea Amalfitano
Journal: Molecular Therapy (2005)

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GSD-II is a recessive lysosomal storage disorder in which lack of human acid-alpha glucosidase(hGAA) activity results in massive accumulations of glycogen in cardiac and skeletal muscles. Recombinant hGAA enzyme replacement therapy(ERT) is currently in clinical trials, but in addition, gene therapy strategies are being explored to serve as an adjunct or alternative to ERT.

We have shown that hepatic targeting of a modified Ad vector expressing hGAA was able to correct the glycogen accumulation in multiple affected muscles in GAA-KO mice, by hepatic secretion of hGAA. In an effort to generate high-level hGAA expression while minimizing vector dosage, we set out to build a replication competent but packaging deficient Ad vector expressing hGAA.

The Ad100K gene occupies about 10% of the entire viral genome confirming its importance in the wt Ad life cycle. The 100K protein is a late viral protein expressed after Ad genome replication. It has many important roles, including transport and trimerization of newly synthesized hexon subunits from the cytoplasm to the nucleus, and acting as a |[ldquo]|scaffolding platform|[rdquo]| for assembly of viral capsids. We hypothesized that an [E1+]Ad vector incorporating deletions within the 100K gene would result in a replication competent but a disabled vector that is unable to express 100K and incapable of hexon trimerization and transport. Previous studies by our group (Hodges et al, 2001) has shown that the deletion of the 100K results in reduced late viral protein production and diminished liver toxicity when an [E1|[minus]|, 100K|[minus]|] vector was administrated in vivo. Based on these previous findings, we hypothesized that a replicating [E1+] Ad vector with a 100K deletion would increase the copy number of the therapeutic vector per target cell after transduction, with diminished toxicity associated with late viral protein production.

We therefore constructed an [E1+, 100K|[minus]|]hGAA Ad vector, and showed that the vector does not induce cytopathic effect when tested in a non-complementing cell line. When tested in vivo, up to 500 times less infectious units (IU) of the replicating vector could achieve similar plasma GAA levels achieved with a non-replicating Ad early after in vivo infection. However, the ratio of IU to viral particle number of this vector was lower than that of most other vectors we have produced. Several possible reasons could contribute to this, including poor transcomplementing ability of our E1 and 100K expressing cell line, or the 100K deletion in this vector is allowing for expression of a dominant negative form of a truncated 100K. To test these possibilities, we are generating new packaging cell lines that express 100K under inducible promoter control, and/or re-engineering the vector as to be fully null for 100K. In optimizing this system we can then further test our hypothesis that uncoupling of Ad viral DNA replication (facilitating increased copy number of the desired transgene encoded by the vector) from late viral protein production via an [E1+, 100K|[minus]|]Ad could result in greater efficacy per Ad vector particle in numerous gene transfer applications.