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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

• the present invention relates generally to the transfer of genes into stem cells, and more specifically to the transfer of genes into human stem cells. Description of the Background Art
• Stem cells such as bone marrow CD34 + CD38 " cells, are highly enriched for pluripotent progenitor cells which account for long term repopulation in vivo (Terstappen et al, 1991,
• hematopoietic progenitor cells have been associated with the induction of an engraftment defect
• CD34 + CD38 cells remain in Go following short term (72-96 hours) in-vitro exposure to various cytokine combinations (Agrawal et al, 1996; Jordan et al, 1996) and these cells also have low or absent expression of cell surface receptors necessary for retro viral transduction (Orlic et al, 1996; Horwitz et al, 1999).
• the lack of cell cycling and the absence of retro viral receptors on human CD34 + CD38 " cells are two major obstacles to successful retroviral based gene therapy using hematopoietic stem cells (Knaan-Shanzer et al, 1995; Miller et al, 1990; Roe et al, 1993; Emmons et al, 1997).
• human CD34 + cells expanded in the PMVEC co-culture system are capable of providing complete hematopoietic reconstitution in a SCID-Hu model (Brandt et al, 1998) and that autologous bone marrow CD34 + cells expanded on PMVEC monolayers are capable of engrafting and hematologically rescuing lethally irradiated baboons (Brandt et al, 1999).
• Fig. 1 shows the cell cycle status of human CD34+CD38- cells at day 0, following 5 days of PMVEC co-culture supplemented with GMCSF+IL-3+IL-6+SCF+Flt-3 ligand and after 5 days of liquid culture supplemented with the identical cytokines.
• Each bar represents the mean percentage of cells in GO, Gl, or G2/S/M phase from 3 separate experiments.
• Fig. 2 shows representative FACS histograms demonstrating the cell cycle status of human CD34+CD38- and CD34+CD38+ cell subsets at day 0 (a), at day 5 of PMVEC co-culture (b), and at day 5 of liquid culture (c). Numbers shown represent the percentage of cells in each quadrant. The lower left quadrant represents cells in GO; upper left, Gl ; upper right G2/S/M phase.
• Fig. 4 shows the phenotype of bone marrow CD34+ cells at day 0 (a) vs. day 5 PMVEC culture (b), and day 5 liquid culture (c).
• CD34+CD38- cells were defined as cells expressing CD34 and lacking CD38 expression compared to the isotype controls.
• Fig. 5 provides a semi-quantitative comparison of RT-PCR product from different human
• the top row reflects the level of GaLVR mR ⁇ A expression
• 2nd row reflects amphoR mR ⁇ A expression
• the bottom row shows GAPDH mR ⁇ A expression in each sample.
• At right is the expression of mR ⁇ A for each transcript in HeLa cells.
• Fig.6(a) through Fig.6(c) are a) Wrights Geimsa stain of human CD34+CD38- cells from 2 experiments following 5 days of PMVEC-co-culture and 3 days of transduction with PG13, b) high magnification view (400x) of HPAP positive CD34+CD38- cells from 2 experiments following expansion in PMVEC co-culture and transduction with PG13, and c) a CFU-GM generated from PMVEC-expanded CD34+CD38- cells which shows HPAP activity following transduction with PG 13.
• hematopoietic stem cells are cells that have not completed the differentiation process.
• the hematopoietic stem cells used in the present invention are adult hematopoietic stem cells.
• the hematopoietic stem cells used in the present invention are hematopoietic, and retain the ability to differentiate into a variety of cell types. Often, these hematopoietic stem cells are contained within and may be harvested from bone marrow.
• One typical class of hematopoietic stem cell that may be expanded and transducted according to the present invention are the CD34 + CD38 " progenator cells.
• CD34 + CD38 " are only a small subset of CD34 + hematopoietic stem cells. Although it is not required when using CD34 + CD38 " cells, CD34 + CD38 " cells may be isolated from CD34 + hematopoietic stem cells before expansion according to the present invention. The method by which the desired hematopoietic stem cells may be harvested and purified is not critical to the instant invention.
• the hematopoietic stem cells are expanded in a culture medium including endothelial cells.
• the culture medium also includes cytokines that stimulate proliferation of hematopoietic stem cells.
• the culture medium may include granulocyte-macrophage colony stimulating factor, interleukin-3, stem cell factor, or interleukin-6, flt3-ligand, and often includes a mixture of thereof.
• the hematopoietic stem cells should be in contact with endothelial cells.
• the endothelial cells are formed as a monolayer.
• the endothelial cells are typically vascular brain cells, such a human brain vascular endothelial cells (HUBEC) or pig microvascular endothelial cells (PMVEC).
• HUBEC is preferable if the transducted cells are to be implanted into human beings.
• Chute et al. United States Patent Application Serial Number 09/452,855, filed December 3, 1999, entitled HUMAN BRAIN ENDOTHELIAL CELLS AND GROWTH MEDR7M AND METHOD FOR EXPANSION OF PRIMITIVE CD34+CD38- BONE MARROW STEM CELLS discusses the composition and characteristics of HUBEC, culture mediums based on HUBEC, and the culture of hematopoietic stem cells in HUBEC and is incorporated herein by reference for all purposes.
• the transduction vectors used can be any of those useful for transduction of the selected class of cells.
• Maloney leukemia virus retroviral vectors such as amph/(MLV) and GaLN pseudotype LAPS ⁇ (GaLV/MLV) viral vectors are useful in the transduction of CD34 + CD38 " cells.
• amph/(MLV) and GaLN pseudotype LAPS ⁇ (GaLV/MLV) viral vectors are useful in the transduction of CD34 + CD38 " cells.
• GaLN pseudotype LAPS ⁇ (GaLV/MLV) viral vectors are useful in the transduction of CD34 + CD38 " cells.
• CD34 + bone marrow progenitor cells were purified by positive immunomagnetic selection by rosetting low density bone marrow mononuclear cells to magnetic beads (Dynal Incorporated, Great Neck, NY) coated with biotinylated anti-CD34 mAb (K6.1). After three to four cycles of magnetic attraction, the beads were disassociated from the cells with an excess of biotin (Gibco, Grand Island, NY) and separated from the cells magnetically and cryopreserved.
• CD34 + cells were thawed rapidly at 37°C, diluted in a 10X volume of pre- warmed (37°C) complete culture medium consisting of Iscove's modified Dulbecco's medium ( DM, Gibco, Grand Island, NY) supplemented with 10% heat-inactivated FCS (Hyclone, Logan, UT) 100 ug/mL L-glutamine (Gibco, Grand Island, NY) and 100 U/mL penicillin/streptomycin (Gibco, Grand IsLand, NY). Unless otherwise noted this culture medium •will be referred to as complete culture medium.
• the thawed CD34 + bone marrow cells were washed twice in complete culture medium, and resuspended at 1 x 10 6 cells/mL. Cell viability was >95% as determined by trypan blue dye exclusion.
• PMVEC were cultured as previously described (Davis et al. 1995). Briefly, PMVEC were plated at cellular concentrations of 1 x 10 s cells/well in gelatin-coated 6-well tissue culture plates (Costar, Cambridge, MA) containing 5 mL of M199 supplemented with 10% heat-inactivated FCS (Hyclone, Logan, UT), 100 mcg mL L-glutamine, 50 mcg/mL heparin, 30 mcg/mL endothelial cell growth factor supplement (Sigma, St. Louis, MO) and 100 mcg/mL penicillin/streptomycin solution.
• the adherent PMVEC monolayers (70-80% confluent) were washed twice with complete culture medium to remove any non-adherent PMVECs and the culture medium was replaced with 7 mL of complete cell culture medium.
• the surface, intracellular, DNA (SID) analysis was performed as described by Jordan et al (Jordan et al. 1996). Briefly, at day 0 and after 5 days of either PMVEC co-culture or liquid culture plus cytokines, CD34 + cells were harvested, washed once, and resuspended in PBS plus 1.0% FCS. Cell surface staining was performed using CD34-APC (Becton-Dickinson, San Jose, CA) and CD38-PE (Becton-Dickinson, San Jose, CA). After surface staining was complete, cells were resuspended in PBS and 0.4% formaldehyde (Electron Microscopy Sciences, E-M grade, Ft. Washington, PA).
• RNA-STAT 60 Tel-Test, Friendswood, TX
• First strand cDNA was synthesized from equalized amounts of mRNA following two reactions with MMLN-RT to ensure one hundred percent incorporation (Stratagene Cloning Systems, LaJolla, CA). cD ⁇ A was amplified with primers for the retroviral
• MJ Research, Inc., Watertown, MA using the following primers: human amphoR (sense primer, 5' CGG AAC ATC TTC GTG GCC TG 3' (SEQ ID NO: 1); anti-sense primer, 5'GCT GGT CAT GAG AGA GCC GTG 3' (SEQ ID NO:2); fragment size, 220bp), human GaLVR (sense primer, 5' GTA GTC CTT CTG AAA GCC CC 3'(SEQ ID NO: 3); anti-sense primer, 5' CAC TGG AGT TTA TTT GGT TGC 3'(SEQ ID NO:4) ; fragment size, 330bp).
• GAPDH PCR was used to estimate experimental cDNA loading as previously described (Blair et al. 1998). Liquid hybridization of PCR products was conducted as described previously (Blair et al. 1998) using the following probes: amphoR (5' ATG GCT CTT CTC ATG TAT GGG 3')(SEQ ID NO:5), GaLVR (5' CCT GCC ACT GTG CCC CTC C 3')(SEQ ID NO: 6), GAPDH (5' TCG CTC CTG GAA GAT GGT GAT GGG ATT 3')(SEQ ID NO:7). Following hybridization, samples were loaded onto 10% acrylamide gels, run at 140V for 3 hr and and then exposed to X-ray film.
• MLV Moloney Leukemia Virus
• amphotropic (ampho/MLV) and GaLV pseudotype LAPSN (GaLV/MLV) viral vectors were generated bearing the human placental alkaline phosphatase (HPAP) reporter gene using PA317 and PG13 packaging cell lines according to methods described previously (Miller et al., 1994; Kiem et al., 1998). Briefly, packaging cell lines were transfected by calcium phosphate method using plasmid pLAPSN (Miller et al, 1994). Transfected packaging cell lines were selected for G418 (neomycin) resistance using 400-800 ug/ml Geneticin (GibcoBRL) in DMEM (Gibco, Grand Island, NY) +10%FCS.
• G418 neomycin
• Producer cell lines were expanded in T225 tissue culture flasks (Falcon, Franklin Lakes, NJ) at 37°C/5% CO 2 using DMEM (Gibco-BRL) with 10% FCS. Collection of virus containing medium (VCM) commenced when packaging cells were about 80% confluent. At this stage the media was replaced with 25ml of DMEM containing 10% FCS and cells were incubated at 32°C. Twenty-four hours later the medium was collected, 0.45uM filtered and frozen at -70°C. VCM was titrated using serial 10 fold dilutions with 8ug/ml polybrene and exposure to HT1080 cells (70-80% confluence) for 24 hours.
• VCM virus containing medium
• Moloney murine leukemia virus vectors encoding for HPAP were prepared using packaging cell lines PA317 and PG13 as described above (Miller et al, 1994; Kiem et al, 1998). Freshly prepared retroviral vector supematants were cryopreserved at -70° C in IMDM + 10% FCS + 1% Penicillin/Streptomycin.
• CD34 + CD38 " and CD34 + CD38 + cells were sorted by FACS and collected for HPAP staining and methylcellulose colony forming assays. Cytospins were prepared from 2-5 x 10 4 CD34 + CD38 " cells and 2-5 x 10 4 CD34 + CD38 + cells and HPAP activity was tested as previously described (Kiem et al. 1998). Briefly, the cytospins were rinsed in PBS, fixed in 2% formaldehyde, rinsed twice in distilled H 2 0, and incubated at 65° C for 30 minutes in order to inactivate endogenous alkaline phosphatase activity.
• the percentage of CD34 + CD38 ⁇ cells which had entered cell cycle was statistically significantly greater in the PMVEC culture group than in the liquid culture group (p ⁇ 0.001).
• a representative FACS histogram showing the percentages of CD34 + CD38 " cells in Go, G ⁇ , and G 2 /S/M phase at day 0, after 5 days of PMVEC co-culture, and following 5 days of liquid culture is shown in Figure 2a-c.
• the CD34 + CD38 " population was not maintained in liquid suspension culture supplemented with the same cytokines.
• Figure 3 shows the results from 3 separate experiments in which the mean starting population of bone marrow cells were 97% ⁇ 2.6 CD34 + and 1.4% + 1.0 CD34 + CD38 " .
• the mean starting population of bone marrow cells were 97% ⁇ 2.6 CD34 + and 1.4% + 1.0 CD34 + CD38 " .
• 37.7% ⁇ 1.7 of the hematopoietic cells remained CD34 + (3a) and the CD34 + CD38 " population had increased to 6.5% + 1.3 of the total population (3b).
• 22.5% + 8.8 of the final population remained CD34 + but the CD34 + CD38 " population decreased to only 0.7% + 0.4.
• the mean level of AmphoR and GaLVR mRNA in day 0 CD34 + CD38 " cells was 5% and 11% of the mean level in HeLa cells, whereas PMVEC-expanded CD34 + CD38 " cells had mean levels which were 16% and 8% of the mean level in HeLa cells.
• the mean levels of AmphoR and GaLVR mRNA in CD34 + CD38 " cells following liquid culture were also low at 1 % and 3%.
• Receptor mRNA levels were normalized to the level of GAPDH mRNA in the same sample and mean levels of mRNA were calculated relative to HeLa cells + SD.
• Purified bone marrow CD34 + cells were expanded in PMVEC co-culture supplemented with GM-CSF + IL-3 + IL-6 + SCF + Flt-3 ligand for 5 days and the expanded population was then transduced for 3 days with high titer (> 1 x 10 5 CFU/mL) MLV vectors bearing the HPAP reporter gene.
• CD34 + cells at day 0 and CD34 + cells which were treated in liquid culture plus the identical cytokines were transduced in an identical fashion with PA317 and PG13 vector supematants.
• PG13 rvPG13
• Data shown represent the mean percentage of cells staining positively for human placental alkaline phosphatase from two separate experiments which tested both vectors (PA 317 and PG 13; titers 1-3 x 10 5 CFU/mL). For each experiment, 5 randomly selected high powered fields (40X) were examined and mean percentages were calculated based upon the number of cells counted per high powered field. Cytospins were prepared from FACS sorted CD34 + CD38 " cells.
• Data shown represent the mean percentage of cells staining positively for human placental alkaline phosphatase from two separate experiments. For each experiment, 5 randomly selected high powered fields (40X) were examined and the mean percentages were calculated based upon the number of cells counted per high powered field. Cytospins were prepared from FACS sorted CD34 + CD38 + cells.
• CD34 + CD38 cells which were collected following 5 days of PMVEC-Co-culture and 3 day exposure to rvPG13 or rvPA317 were then placed in methylcellulose to assess for HPAP expression in 14 day colony forming cells (CFC). At day 14, 35.1% ⁇ 15.5 of the CFC derived from CD34 + CD38 " cells transduced with rvPA317 were HPAP positive. Similarly, 38.1 % ⁇ 17.5 of the CFC derived from CD34 + CD38 " cells exposed to rvPG13 demonstrated HPAP positivity (Table 4A). A representative CFU-GM showing HPAP expression is shown in Figure 6c.
• CD34 + CD38 cells at day 0 and following liquid culture were also transduced with rvPG13 and rvPA317 for 3 days and then placed in methylcellulose colony forming assays. The mean number of transduced colonies in both the day 0 and liquid culture groups were signgificantly lower than the PMVEC co-culture group (Table 4).
• Data shown represent the mean percentage of colonies staining positively for human placental alkaline phosphatase from two separate experiments which tested both vectors (PA 317 and PG 13; titers 1-3 x 10 5 CFU/mL). For each experiment, triplicate methylcellulose cultures were examined and mean percentages were calculated based upon the number of colonies counted per each dish. Methylcellulose CFC cultures were prepared from FACS sorted CD34 + CD38 " cells.
• the CD34 + CD38 " population within human bone marrow is enriched for cells with in vivo long term repopulating capacity (Bhatia et al, 1997; Berardi et al, 1995; Hao et al, 1995) and therefore, these cells are ideal targets for therapeutic gene transfer.
• the efficiency of retroviral gene transfer into these cells has been shown to be consistently low (Landsdorp et al, 1993; Dao et al, 1998; McCowage et al, 1998).
• PMVEC co-culture causes a very high percentage (>93%) of the CD34 + CD38 " population to enter cell division after only a short (5 day) period of co-culture.
• >95% of the day 0 CD34 + CD38 " cells harvested from the bone marrow reside in G 0 and these cells could not be efficiently transduced using the same transduction methods.
• the level of cell cycling in hematopoietic stem cells has been consistently associated with a higher efficiency of retroviral gene transfer in previous studies (Knaan-Shanzer et al, 1995; Miller et al, 1990).
• PMVEC monolayers supplemented with IL-3 + IL-6 + GMCSF + SCF + Flt-3 ligand offer a specific advantage for gene transfer applications by promoting a high level of cell cycling in CD34 + CD38 " cells in short-term culture while maintaining a population of cells capable of in vivo engraftment. Therefore, expansion of human CD34 + CD38 " cells on PMVEC monolayers followed by retroviral transduction should increase the likelihood that gene marked cells will give rise to long term repopulation in vivo.
• retroviruses are capable of infecting target cells through receptors other than the identified envelope receptors (e.g. amphoR or GaLVR) (Miller et ah, 1996), so unknown receptor changes which occur during PMVEC co-culture also may have accounted for the increased transduction efficiency we have observed.
• envelope receptors e.g. amphoR or GaLVR
• cord blood CD34 + cells may be due, in part, to significant ontogenic differences between bone marrow CD34 + cells and umbilical cord blood CD34 + cells with regard to both the frequency of primitive cells and their proliferative potential as well as their expression of retroviral receptors (Orlic et ah, 1998; Zandstra et ah, 1998; Weeks et ah, 1998). Nonetheless, autologous bone marrow CD34 + cells will continue to be a primary and preferred source of hematopoietic stem cells for human gene therapy applications.
• CTLA-4 Ligation delivers a unique Signal to Resting CD4 T Cells that inhibits Interleukin-2 secretion but allows Bcl-xL Induction. J. Immunol. 160, 12-16.
• Porcine brain microvascular endothelial cells support the in vitro expansion of human primitive hematopoietic bone marrow progenitor cells with a high replating potential: requirement for cell-to-cell interactions and colony-stimulating factors. Blood 85, 1751-1761.
• Retroviral gene transduction of adult peripheral blood or marrow derived CD34+ cells for six hours without growth factors or on autologous stroma does not improve marking efficiency assessed in vivo. Blood 89, 4040-4046.
• Granulocyte colony stimulating factor mobilized peripheral blood stem cells enter into Gl of the cell cycle and express higher levels of amphotropic retrovirus receptor mRNA. Exp Hematol 27, 1160-1167.
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• KNAAN-SHANZER S., VALERIO, D., VAN BEUSECHEM, V.W. (1995).
• MILLER AD. (1996). Cell-surface receptors for retroviruses and implications for gene transfer. Proc Natl Acad Sci USA 93, 11407-11413. MILLER, D.G., ADAM, M.A., MILLER, A.D. (1990). Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 10, 4239- 4242.
• MILLER D.G., EDWARDS, R.H., MILLER, A.D. (1994). Cloning of the cellular receptor for amphotropic murine retroviruses reveals homoloogy to that for gibbon ape leukemia virus. Proc Natl Acad Sci USA 91, 78-82.
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• E -3 interleukin-3
• IL-6 interleukin-6
• IL-11 stem cell factor
• Flt-3 ligand induces proliferation of quiescent human bone marrow CD34+CD38- cells and maintains progenitor cells in vitro. Blood 87, 3563-3570.
• the murine stromal cell line AFT024 acts specifically on human CD34 + CD38- progenitors to maintain primitive function and immunophenotype in vitro. Exp Hematol 26, 612-619.
• VAN HENNIK P., VERSTEGEN, M., BIERHUIZEN, M. LIMON, A., WOGNUM, A., CANCELAS, J., BARQUINERO, J., PLOEMACHER, R., WAGEMAKER, G. (1998). Highly efficient transduction of the green fluorescent protein gene in human umbilical cord blood stem cells capable of cobblestone formation in long term cultures and multilineage engraftment of immunodeficient mice. Blood 92, 4013-4022. VARAS, F., BERNARD, A., BUEREN, J.A. (1998). Restrictions in the stem cell function of murine bone marrow grafts after ex vivo expansion of short term repopulating progenitors. Exp Hematol 26, 100-109.
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