Multi-Copy Integration after HIV-Derived Lentivirus Vector Transduction of Murine Hematopoietic Stem Cells Does Not Promote Clonal Proliferation in Primary or Secondary Recipients.

Replication competent Moloney murine leukemia virus (MLV) retrovirus is used experimentally in murine models to identify potential oncogenes by repeat infection and resultant insertion of multiple proviral copies into the target cell genome. Mutagenicity in this system relies on proviral insertion in proximity of oncogenes, or their regulatory sequences leading to subsequent activation and clonal expansion. Replication incompetent MLV-derived vector particles are attractive vehicles for gene therapy strategies, and have been shown to retain these mutagenic properties after transduction of highly enriched murine and human hematopoietic progenitor and stem cells. Work by others suggests that this may in part occur in a vector particle dose and copy number dependent, transgene independent fashion (Modlich et al., Blood 2003 Suppl., abstract 699; Brugman et al., Molecular Therapy 2004 Suppl., abstract 1046).

It is unclear if the same properties of mutagenic potential and copy number dependence apply to the insertion of HIV-derived lentivirus vectors. To address this question we have used VSV-G pseudotyped lentiviral vector (VSV-G/RRLsin-cPPThPGK-EGFPwpre) particles to transduce non-enriched mouse hematopoietic stem cells at escalating vector particle concentrations for subsequent repopulation of myeloablated murine recipients. Cells were transduced in culture with a single round of infection at a multiplicity of 1, 3, 10, or 30 in the presence of multiple cytokines and fibronectin fragment. Recipient animals readily reconstituted their hematopoietic system and demonstrated GFP marking in myeloid, B- and T- lymphoid cells. Peripheral blood counts, kinetics of GFP marking and flow-cytometric light scatter profiles showed no evidence of clonal proliferation. Primary recipients (n=23) were sacrificed between 5 and 7 months from transplantation to examine their marrow, peripheral blood and (in part) spleen for proviral marking by flow cytometry and real-time PCR. Results show a predicted increase in average proviral copy number ranging from 1.8 (range 1.2 – 2.8) after low MOI (1) infection to 17.5 (range 2.9 – 46.8) after high MOI (30) infection. Further, PCR amplification of genomic-proviral junction sites from progenitor colonies obtained at sacrifice of primary recipients showed no evidence of clonal restriction in the cohort of animals with an average of 7.9 proviral copies per GFP-marked cell (MOI 10 cohort). Secondary recipients (n=71) in all dose groups were followed for gene marking and, to date, have shown no signs of vector-driven clonal evolution.

Our results suggest that HIV-derived lentivirus vector does not appear to have the same mutagenic properties at similar genomic copy numbers that others report for MLV-derived vectors in a comparable murine model system. This observation is consistent with distinct genomic insertion site preferences reported for HIV-derived vectors. We herein propose that, based on these and other advantageous features, lentivirus vectors are ideally suited for further pre-clinical and clinical exploration.