Patrick F. Kelly

St. Jude Children's Research Hospital, Memphis, Tennessee, United States

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Publications (11)49.39 Total impact

  • Patrick F. Kelly · Jody Carrington · Amit Nathwani · Elio F. Vanin ·
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    ABSTRACT: Limited functional expression of the viral envelope receptor is a recognized barrier to efficient oncoretroviral mediated gene transfer. To circumvent this barrier we evaluated a number of envelope proteins with respect to gene transfer efficiency into primitive human hematopoietic stem cell populations. We observed that oncoretroviral vectors pseudotyped with the envelope protein of feline endogenous virus (RD114) could efficiently transduce human repopulating cells capable of establishing multilineage hematopoiesis in immunodeficient mice after a single exposure to RD114-pseudotyped vector. Comparable rates of gene transfer with amphotropic and GALV-pseudotyped vectors have been reported, but only after multiple exposures to the viral supernatant. Oncoretroviral vectors pseudotyped with the RD114 or the amphotropic envelopes had similar stability in vitro, indicating that the increased efficiency in gene transfer is at the receptor level likely due to increased receptor expression or an increased receptor affinity for the RD114 envelope. We also found that RD114-pseudotype vectors can be efficiently concentrated, thereby removing any adverse effects of the conditioned media to the long-term repopulating potential of the target human hematopoietic stem cell. These studies demonstrate the potential of RD114-pseudotyped vectors for clinical use.
    Annals of the New York Academy of Sciences 06/2006; 938(1):262-277. DOI:10.1111/j.1749-6632.2001.tb03596.x · 4.38 Impact Factor
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    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.
    Molecular Therapy 08/2005; 11. DOI:10.1016/j.ymthe.2005.06.390 · 6.23 Impact Factor
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    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.
    Blood 11/2003; 102(8):2789-97. DOI:10.1182/blood-2002-05-1482 · 10.45 Impact Factor
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    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.
    Blood Cells Molecules and Diseases 01/2003; 30(1):132-43. DOI:10.1016/S1079-9796(03)00005-6 · 2.65 Impact Factor
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    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.
    Molecular Therapy 04/2002; 5(3):242-51. DOI:10.1006/mthe.2002.0549 · 6.23 Impact Factor
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    ABSTRACT: Oncoretrovirus vectors pseudotyped with the feline endogenous retrovirus (RD114) envelope protein produced by the FLYRD18 packaging cell line have previously been shown to transduce human hematopoietic progenitor cells with a greater efficiency than similar amphotropic envelope-pseudotyped vectors. In this report, we describe the production and efficient concentration of RD114-pseudotyped murine leukemia virus (MLV)-based vectors. Following a single round of centrifugation, vector supernatants were concentrated approximately 200-fold with a 50 to 70% yield. Concentrated vector stocks transduced prestimulated human CD34+ (hCD34+) cells with approximately 69% efficiency (n = 7, standard deviation = 4.4%) using a single addition of vector at a low multiplicity of infection (MOI = 5). Introduction of transduced hCD34+ cells into irradiated NOD/SCID recipients resulted in multilineage engraftment with long-term transgene expression. These data demonstrate that RD114-pseudotyped MLV-based vectors can be efficiently concentrated to high titers and that hCD34+cells transduced with concentrated vector stocks retain in vivo repopulating potential. These results highlight the potential of RD114-pseudotyped oncoretrovirus vectors for future clinical implementation in hematopoietic stem cell gene transfer.
    Journal of Virology 11/2001; 75(20):9995-9. DOI:10.1128/JVI.75.20.9995-9999.2001 · 4.44 Impact Factor
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    ABSTRACT: Human hematopoietic cells with in vivo repopulating potential hold much promise as a target for corrective gene transfer for numerous inherited or acquired hematopoietic disorders. Here we demonstrate long-term hematopoietic reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice with human CD34(+) cells transduced by an HIV-1-based self-inactivating (SIN) vector encoding the enhanced green fluorescent protein (EGFP). Human umbilical cord CD34(+) cells were transduced (up to 76%) at a low multiplicity of infection (MOI of 5) in the absence of cytokine prestimulation. Introduction of transduced hCD34(+) cells into irradiated recipients resulted in multilineage engraftment and stable transgene expression for 18 weeks posttransplantation. Bone marrow from transplanted mice contained up to 50% hCD45(+) cells and up to 63% hCD45(+)/EGFP(+) cells. Analysis of extramedullar splenic reconstitution showed up to 13% hCD45(+) cells and up to 41% hCD45(+)/EGFP(+) cells. Analysis of human progenitor cells isolated from bone marrow of recipient animals showed equivalent percentages of EGFP(+) colony-forming cells (CFCs) by fluorescence microscopy and by PCR analysis of provirus sequences, indicating minimal transgene silencing in vivo. These findings demonstrate the utility of lentivirus-based SIN vectors for hematopoietic stem cell gene transfer and provide strong support for their future clinical evaluation.
    Human Gene Therapy 07/2001; 12(9):1079-89. DOI:10.1089/104303401750214294 · 3.76 Impact Factor
  • Kevin D. Bunting · Taihe Lu · Patrick F. Kelly · Brian P. Sorrentino ·
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    ABSTRACT: Janus kinase 3 (JAK3) is an essential component of cytokine receptor signal transduction pathways required for normal lymphocyte development and function. JAK3 deficiency in both mice and humans results in severe combined immunodeficiency (SCID) and increased susceptibility to opportunistic infections. We have previously shown that JAK3 gene transfer into irradiated recipients could restore immune function. However, since this toxic conditioning would be undesirable for infants in a clinical application, we have tested whether immune function could be restored in nonmyeloablated JAK3-deficient (-/-) mice. Murine JAK3 retroviral vectors were transduced into hematopoietic stem cells from the livers of newborn JAK3(-/-) mice. These cells were then injected intraperitoneally into nonirradiated JAK3(-/-) neonates. Transduced cells were detectable in these mice at time points 4 to 6 months after injection and resulted in significant correction of T and B lymphocyte numbers and circulating immunoglobulin (Ig) levels. After immune challenge with a dose of influenza A virus that was lethal to nonmanipulated JAK3(-/-) mice, mice injected with transduced cells showed development of circulating virus-specific IgG and enhanced survival. This work shows that the large selective advantage for JAK3-corrected lymphoid cells may be sufficient to overcome the need for myeloablative conditioning in JAK3 gene therapy protocols.
    Human Gene Therapy 12/2000; 11(17):2353-64. DOI:10.1089/104303400750038462 · 3.76 Impact Factor
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    ABSTRACT: Bone marrow stromal cells (MSCs) are unique mesenchymal cells that have been utilized as vehicles for the delivery of therapeutic proteins in gene therapy protocols. However, there are several unresolved issues regarding their potential therapeutic applications. These include low transduction efficiency, attenuation of transgene expression, and the technical problems associated with drug-based selection markers. To address these issues, we have developed a transduction protocol that yields high-level gene transfer into human MSCs, employing a murine stem cell virus-based bicistronic vector containing the green fluorescent protein (GFP) gene as a selectable marker. Transduction of MSCs plated at low density for 6 hr per day for 3 days with high-titer viral supernatant resulted in a gene transfer efficiency of 80+/-6% (n = 10) as measured by GFP fluorescence. Neither centrifugation nor phosphate depletion increased transduction efficiency. Assessment of amphotropic receptor (Pit-2) expression by RT-PCR demonstrated that all MSCs expressing the receptor were successfully transduced. Cell cycle distribution profiles measured by propidium iodide staining showed no correlation with the susceptibility of MSCs to transduction by the retroviral vector. Human MSCs sequentially transduced with an adenoviral vector encoding the ecotropic receptor and ecotropic retroviral vector encoding GFP demonstrated that all MSCs are susceptible to retroviral transduction. We further showed that both genes of bicistronic vector are expressed for at least 6 months in vitro and that transgene expression did not affect the growth or osteogenic differentiation potential of MSCs. Future studies will be directed toward the development of gene therapy protocols employing this strategy.
    Human Gene Therapy 06/1999; 10(7):1163-73. DOI:10.1089/10430349950018157 · 3.76 Impact Factor
  • Jay T. Evans · Patrick F. Kelly · Eduardo O'Neill · J. Victor Garcia ·
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    ABSTRACT: The efficient transfer and sustained expression of a transgene in human hematopoietic cells with in vivo repopulating potential would provide a significant advancement in the development of protocols for the treatment of hematopoietic diseases. Recent advances in the ability to purify and culture hematopoietic cells with the CD34+CD38- phenotype and with in vivo repopulating potential from human umbilical cord blood provide a direct means of testing the ability of transfer vectors to transduce these cells. Here we demonstrate the efficient transduction and expression of enhanced green fluorescent protein (EGFP) in human umbilical cord-derived CD34+CD38- cells, without prestimulation, using a lentivirus-based gene transfer system. Transduced CD34+CD38- cells cultured in serum-free medium supplemented with SCF, Flt-3, IL-3, and IL-6 maintained their surface phenotype for 5 days and expressed readily detectable levels of the transgene. The average transduction efficiency of the CD34+CD38- cells was 59 +/- 7% as determined by flow cytometry. Erythroid and myeloid colonies derived from transduced CD34+CD38- cells were EGFP positive at a high frequency (66 +/- 9%). In contrast, a murine leukemia virus-based vector transduced the CD34+CD38- cells at a low frequency (<4%). These results demonstrate the utility of lentiviral-based gene transfer vectors in the transduction of primitive human hematopoietic CD34+CD38- cells.
    Human Gene Therapy 06/1999; 10(9):1479-89. DOI:10.1089/10430349950017815 · 3.76 Impact Factor
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Publication Stats

449 Citations
49.39 Total Impact Points


  • 2001-2006
    • St. Jude Children's Research Hospital
      • • Division of Experimental Hematology
      • • Department of Hematology
      Memphis, Tennessee, United States
    • University of Texas Southwestern Medical Center
      • Department of Internal Medicine
      Dallas, Texas, United States