764. Dosing and Re-Administration of Intravenous Lentiviral Vector for Liver-Directed Gene Transfer in Young Rhesus Monkeys and ADA-Deficient Mice
Authors: Denise A. Carbonaro-Sarracino, Alice F. Tarantal, Chang I. Lee, Michael L. Kaufman, Michele Martinez, Danielle N. Clark, Colin Koziol, Donald B. Kohn
Journal: Molecular Therapy (2016)

Liver-directed gene transfer with lentiviral vectors (LV) is a treatment modality being investigated for a number of genetic diseases. To study re-administration of LV, rhesus monkeys and Adenosine deaminase (ADA) knockout mice were repeatedly dosed with vesicular stomatitis virus (VSV) pseudotyped LV. In some studies the LV were modified with polyethylene glycol (PEG) since others showed that PEG modification of lysine residues of VSV increases LV half-life in serum and reduces compliment-mediated inactivation. ADA-deficiency is a defect in purine metabolism that results in severe combined immunodeficiency due to substrate accumulation. Unlike humans, ADA-/- mice do not survive to day 21 if not treated, however, when treated with intravenous ADA LV, high ADA expression in the liver was associated with prolonged survival and immune reconstitution. Liver vector copy number (VCN) was determined by qPCR. Anti-vector IgG concentration in plasma was determined by ELISA, using VSV LV as the capture antigen and a mouse anti-VSV IgG standard. Untreated animals have 10 µg/ml of reactive IgG. Direct competitive ELISA was used to determine the PEG concentration on PEG LV. ADA-/- mice treated with 5×10e10TU/kg of ADA LV at birth did not develop anti-vector antibodies (n=12) and showed 0.3±0.23 liver VCN whereas ADA-/- mice immune reconstituted with ERT and treated with 1.5×10e10 TU/kg at 4 months (m) produced 100-fold more anti-vector IgG (n=5; 6890±1143µg/ml; p=0.01) with no difference in VCN when adjusted for dose. Similar results were found in rhesus monkeys treated with 2.0×10e9 TU/kg of LV at 1 month postnatal age (n=2; 4030±10µg/ml) compared to rhesus monkeys treated at 4 m postnatal age (n=2; 17800±300µg/ml). In immune competent ADA+/- mice treated with ADA LV at birth followed by unmodified enhanced green fluorescent protein (eGFP) LV (n=3; 0.05 VCN; 1390µg/ml) or PEG modified eGFP LV (n=3; 0.05 VCN; 1330µg/ml) at 8 m, the liver VCN and the anti-vector responses were not different. However, if mice were treated with an unmodified eGFP LV or PEG modified eGFP LV at 4 m and an unmodified ADA LV at 8 m, anti-vector IgG concentration was increased 10-fold with the unmodified eGFP LV (n=3; 114666±666µg/ml) compared to the PEG modified eGFP LV (n=3; 9733±333µg/ml); however, the second LV was inactivated and no vector was detected from mice in either group. When ADA+/- mice were treated with an unmodified ADA LV at birth and again at 5 m (booster), followed by an unmodified (n=5) or PEG modified (n=5) eGFP LV at 8 m, the third injection was associated with acute toxicity and reduced probability of survival in both groups (40%; p=0.01). In immune competent rhesus monkeys dosed with an unmodified LV carrying a non-expressed transgene at 3 m of age and followed by an unmodified LV or a PEG modified LV carrying a different non-expressed transgene at 6 m, the second vector was not detected in either group and both the unmodified LV (n=2; 15600±1,200µg/ml) and the PEG-modified LV (n=2; 15500±900µg/ml) had similar anti-vector responses. In conclusion, LV dosing at birth is associated with reduced anti-vector responses compared to dosing at ~3 m of age. Repeat LV dosing can result in inactivation of a second vector administration that is associated with higher anti-vector IgG responses and a higher probability of acute toxicity when dosed months apart. PEG modification does not appear to protect the second dose from inactivation or prevent toxicity.