[Show abstract][Hide abstract] ABSTRACT: Replication defective retroviruses are attractive tools for hematopoietic stem cell (HSC) gene therapy because of their ability to integrate into genomic DNA. Reports of neoplasia related to vector insertional proto-oncogene activation have raised safety concerns, however in these instances the vector itself carried a potentially cooperating transforming gene. We report for the first time a replication-defective vector-associated neoplasia in a non-human primate. Six years after autologous transplantation with G-CSF/SCF mobilized CD34+ cells transduced with a MSCV RD114 pseudotyped retroviral vector expressing eGFP and a drug resistant variant of the human dihydrofolate reductase gene (L22Y), a rhesus macaque developed a fatal myeloid sarcoma. Of note, this animal had high level transient marking during the first year following transplantation derived from a single transduced clone containing two vector insertions, over the next five years 1-3% GFP+ blood cells and was clinically well (Kelly et al, 2003). The animal received one cycle of trimextrexate and NBMP with transient increase in GFP+ blood cells 2001 (Persons et al, 2004). In 9/04 the animal developed abdominal distress and had a renal mass. Blood showed 30% GFP+ granulocytes, but no leukemia. Following surgery the animal died of pancreatitis and sepsis. Necroscopy revealed myelomonocytic tumor cell infiltrates with large round nuclei and variable amount of cytoplasm (MPO+, CD45+/-, CD68+, CD3-, CD20-, κ-/λ-, GFP+/-) in both kidneys, liver, pancreas, spleen, lymph nodes and in the choroid plexus. Taqman demonstrated a vector copy number of at least 1/cell in the tumor, suggesting association with vector. There was no detectable replication-competent helper virus in the tumor or the blood, based on PCR for helper gag/pol sequences. Blood cells and enriched tumor cells (>95%) have been analyzed via LAM-PCR identification of integration sites. A vector insertion in the first intron of the BCL2-A1 gene was found in the tumor, along with a second valid integration site that could not be mapped unequivocally using the human genome. PCR analysis using primers specific for the BCL2-A1 insertion suggested very high level contribution from the clone in the first year following transplantation, which then became undetectable and increased in the blood concurrent with the development of the myeloid sarcoma. Thus a single clone containing the vector insertion into the BCL2-A1 gene made a major multilineage contribution to hematopoieses during the first six months after reconstitution, became dormant and then re-emerged as a myeloid neoplasm after six years. The BCL2-A1 protein blocks apoptosis and is associated with a poor prognosis in human acute myeloid leukemia. Further research is required for risk assessment of current available vector systems, especially when targeting long-lived HSC.
[Show abstract][Hide abstract] ABSTRACT: In previous studies amphotropic MFGS-gp91phox (murine onco-retrovirus vector) was used in a clinical trial of X-linked chronic granulomatous disease (X-CGD) gene therapy to achieve transient correction of oxidase activity in 0.1% of neutrophils. We later showed that transduced CD34+ peripheral blood stem cells (CD34+ PBSCs) from this trial transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in correction of only 2.5% of human neutrophils. However, higher rates of transduction into stem cells are required. In the current study we demonstrate that the same vector (MFGS-gp91phox) pseudo-typed with RD114 envelope in a 4-day culture/transduction regimen results in a 7-fold increase in correction of NOD/SCID mouse repopulating X-CGD CD34+ PBSCs (14%-22% corrected human neutrophils; human cell engraftment 13%-67%). This increase may result from high expression of receptor for RD114 that we demonstrate on CD34+CD38- stem cells. Using RD114-MFGS encoding cyan fluorescent protein to allow similar studies of normal CD34+ PBSCs, we show that progressively higher levels of gene marking of human neutrophils (67%-77%) can be achieved by prolongation of culture/transduction to 6 days, but with lower rates of human cell engraftment. Our data demonstrate the highest reported level of functional correction of any inherited metabolic disorder in human cells in vivo with the NOD/SCID mouse system using onco-retrovirus vector.
[Show abstract][Hide abstract] ABSTRACT: The ability to efficiently transfer a gene into repopulating hematopoietic stem cells would create many therapeutic opportunities. We have evaluated the ability of particles bearing an alternative envelope protein, that of the feline endogenous virus (RD114), to transduce stem cells in a nonhuman primate autologous transplantation model using rhesus macaques. We have previously shown this pseudotyped vector to be superior to the amphotropic vector at transducing cells in umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice. Gene transfer efficiency as reflected by the number of genetically modified cells in hematopoietic tissues varied among the five monkeys studied from low levels (<1%) in three animals to much higher levels in two (20-60%). An animal that exhibited extremely high levels for several weeks was found by vector genome insertion site analysis to have reconstitution predominantly with a single clone of cells. This variability among animals is in keeping with computer simulations of reconstitution with limiting numbers of stem cells genetically modified at about 10% efficiency. Our studies provide insights into the biology of hematopoietic reconstitution and suggest approaches for increasing stem cell targeted gene transfer efficiency.
[Show abstract][Hide abstract] ABSTRACT: Substantial effort has been invested in developing methodologies for efficient gene transfer into human, repopulating, hematopoietic stem cells. Oncoretroviral vectors are limited by the lack of nuclear mitosis in quiescent stem cells during ex vivo transduction, whereas the preintegration complex of lentiviral vectors contains nuclear-localizing signals that permit genome integration without mitosis. We have developed a flexible and versatile system for generating lentiviral vector particles and have pseudotyped such particles with amphotropic, ecotropic, feline endogenous virus (RD114) or vesicular stomatitis virus (VSV-G) envelope proteins. Particles of all four types could be concentrated approximately 100-fold by ultracentrifugation or ultrafiltration. RD114 or amphotropic particles were more efficient than VSV-G-pseudotyped particles at transducing human cord blood CD34(+) cells and clonogenic progenitors within that population. Amphotropic particles transduced cytokine-mobilized, human peripheral blood CD34(+) cells capable of establishing hematopoiesis in immunodeficient mice more efficiently than the other two types of particles. We conclude that the use of amphotropic pseudotyped lentiviral vector particles rather than the commonly used VSV-G-pseudotyped particles should be considered in potential applications of lentiviral vectors for gene transfer into this therapeutically relevant target cell population.