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  • 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
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/16045—Special targeting system for viral vectors

Definitions

• the present invention relates to the field of recombmant retroviral particles, to retroviral gene delivery vehicles.
• compositions for transduction as well as pharmaceutical compositions for the treatment of disorders with a genetic component.
• the invention provides in one of its embodiments a method of gene transfer into e.g. plu ⁇ potent hematopoietic stem cells and their descendants, enabling successful transduction of 90% of CD34+ cells, including transplantable cell populations comprising hematopoietic stem cells that give rise to progeny expressing the transduced gene(s) .
• the use of the method e.g. includes and is included m a method for treatment of a variety of hereditary and acquired human diseases by transfer of therapeutically active genes into hematopoietic stem cells and genetic marking of suchs cells.
• the hematopoietic system produces perpetually large numbers of blood cells, which have a limited life span and need to be perpetually renewed throughout the life of a mammal. This renewal is maintained through proliferation and differentiation of a small number of hematopoietic stem cells in the bone marrow.
• the definition of stem cells is not always clear within the art.
• a functional definition is used, which defines stem cells as those cells capable of (long term) reconstitution of a hematopoietic system. This definition is often felt to include at least some early progenitor cells.
• hematopoietic stem cells are a highly suitable target for gene therapy for a variety of hereditary and acquired diseases within and outside the hematopoietic system.
• retrovirus mediated gene transfer has met with only limited success due to the difficulty of obtaining sufficient numbers of successfully transduced, transplantable, long-term repopulating hematopoietic stem cells .
• the invention provides a method for transducing target cells with gene delivery vehicles of retroviral origin comprising providing a selection of CD34 positive cells from a culture of cells, taking a plurality of samples of said CD34 positive population, diluting and/or concentrating said samples to provide a range of concentrations of cells per volume, contacting samples m said range with a composition comprising said gene delivery vehicles and determining the optimal concentration of target cells for efficient transduction and diluting or concentrating said CD34 positive population to said optimal concentration and contacting said population with said gene delivery vehicles to allow for said transduction of target cells.
• the population of CD34 positive cells such as they can be found in e.g. umbilical cord blood or bone marrow includes the stem cells as defined herein above (i.e. the cells capable of long term repopulation) .
• stem cells as defined herein above (i.e. the cells capable of long term repopulation) .
• a very important variable in the efficiency of transduction is a ratio between number of cells (or cell concentration) and the number of transducing particles. According to our invention these should be optimized vis a vis one another, which may sometimes lead to increasing particle titers and/or target cell concentrations, but surprisingly also to decreasing viral particles and/or lowering cell concentrations. Therefore a range of concentrations should be tried with samples from the target population of cells.
• Gene delivery vehicles of retroviral origin are all vehicles comprising genetic material and/or protemaceous material derived from retroviruses. Typically the most important features of such vehicles are the integration of their genetic material into the genome of a target cell and their capability to transduce stem cells. These elements are deemed essential in a functional manner, meaning that the sequences need not be identical to retroviral sequences as long as the essential functions are present.
• the methods of the invention are however especially suitable for recombinant retroviral particles, which have most if not all of the replication and reproduction features of a retrovirus, typically m combination with a producer cell having some complementing elements. Normally the retroviral particles making up the gene delivery vehicle are replication defective on their own.
• the invention is particularly suited for the production of gene delivery vehicles, however other retroviral particles can also be produced according to the invention.
• the concentration of target cells is optimized, but also the concentration of virus.
• optimization of all concentrations involved m binding or interaction is preferred.
• initial titers are preferred.
• Methods to arrive at those are also provided by the present invention.
• the invention further provides a method wherein target cell concentrations are optimized further comprising optimizing the concentration of said gene delivery vehicles for optimal transduction efficiency. It is of course that a gene delivery vehicle is intended to read on any vehicle capable of delivering genetic material to a target cell, whether the genetic material is actually a gene, an antisense molecule or a cosuppressive nucleic acid (encoding molecule), etc.
• nucleic acids to be provided to target cells are well known m the art and include such molecules as to replace inborn errors/deficiencies of the hematopoietic system, which may include hemoglobin genes and their regulatory elements for the thalassemia ' s and sickle cell anemia's and sequences to repair the various forms of severe combined immunodeficiency, such as caused by adenosine deammase deficiency and that known as severe X linked immunodeficiency, or genes encoding enzymes for diseases known as lysosomal storage diseases, such as Hurler's,
• the invention also provides a method as described above wherein said target cells are cultured the presence of fibronectm, retronectm or a functional equivalent thereof, preferably further comprising optimizing the concentration of said fibronectm, retronectm or said functional equivalent for optimal transduction efficiency.
• the target cells of the present invention comprise populations of CD34 positive cells, which are efficiently transduced by retroviral particles, preferred are those populations wherein said CD34 positive cells comprise umbilical cord blood cells or bone marrow cells.
• CD34 positive cells comprise umbilical cord blood cells or bone marrow cells.
• the invention also provides a method as described above wherein a composition of retroviral gene delivery vehicles of improved titer is applied.
• a method for improving the virus titer is applied which involves improving the producer cell line.
• the invention also provides a method wherein said virus titer is improved by providing a culture of producer cells of a retroviral gene delivery vehicle, subclon g said culture of producer cells, culturmg the resulting subclones and selecting the clones producing the highest virus titers, possibly based on multiple copies of the provirus due to reinfection.
• a number of cells from established producer cell lines loose some of their ability to produce effective retroviral particles.
• Subclonmg appears to be a way to select for those cells retaining that ability. Other ways of selecting for such cells are also included the present invention.
• the method of improving virus titers can also be used apart from the improvement of transduction.
• the invention also provides a method for producing retroviral particles at high titers, comprising providing a culture of producer cells producing retroviral particles, subclonmg said culture of producer cells, culturmg the resulting subclones and selecting the cultures producing the highest virus titers.
• the invention is preferably applied to gene delivery vehicle production.
• the invention provides in yet another embodiment a method as ⁇ ust described wherein said retroviral particles are gene delivery vehicles.
• producer cells are well known in the art.
• the preferred ones are mouse fibroblast cells, originating from PG13 which is pseudotyped with the gibbon ape leukemia virus (GALV) .
• GA V-receptors GLV-1 or P ⁇ t-1) are present on human hematopoietic cells.
• compositions obtainable by the methods of the invention.
• compositions comprising retroviral particles at high titer obtainable by a method as disclosed above, preferably those wherein said retroviral particles are gene delivery vehicles.
• retroviral particles capable of transducing hematopoietic stem cells and/or progenitor cells, preferably wherein said retroviral particles are capable of transducing umbilical cord blood cells and/or bone marrow cells
• the invention also provides the pharmaceutical use of these compositions, particularly m the treatment of diseases having a genetic component, such as the various genetic hemoglobin orders, the large group of rare diseases collectively known as severe combined immune deficiencies, the group of lysosomal storage diseases, especially with a strong hematopoietic and/or visceral expression, such as Gaucher's disease, but also possibly Krabbe ' s disease, as well as in the treatment of infectious disease, notably HIV infection, or cancer.
• a composition comprising retroviral particles will involve the transduction of CD34 positive target cells. Such transduced cells are typically made in vitro and are also part of the present invention.
• the invention provides a composition for the treatment of a hereditary disease or a pathological condition related to a genetic defect or a genetic aberration, comprising a plurality of CD34 positive cells transduced with a composition of retroviral particles according to the invention, or a composition for the treatment of a hereditary disease or a pathological condition related to a genetic defect or a genetic aberration, comprising a plurality of CD34 positive cells, said composition being obtainable by a method according to the invention.
• the object of the present invention is to provide a generally applicable method for retrovirus mediated transfer of therapeutic and marker genes into plu ⁇ potent hematopoietic stem cells.
• the invention includes the unexpected and surprising finding that transduction is dependent on the concentration of target cells during the transduction period and that selection of (high titer) subclones of a by itself effective producer cell line decreases this dependence on cell concentration.
• EGFP transduced CD34+ cells were transplanted into immunodeficient mice irradiated with 3.5 Gy (g-rays total body irradiation. The mice were sacrificed at day 35 to measure content of repopulating cells and to assess the multilineage nature of the transduced cells by flow cytometry. Typically, the optimized procedure resulted in percentages of up to 80% EGFP expression (Table 1) which was multilineage in nature ( Figure 4) .
• the pseudotyped retroviral producer cell line PG13/EGFP7 was kindly provided by J. Barquinero (Institut de Recerca Oncologica, Barcelona, Spain) .
• the cell line was developed by transducing the PG13 packaging cell line (kindly provided by D. Miller, Fred Hutchinson Cancer Research Centre, Seattle, WA) with 0,45 ⁇ m filtered supernatant from PA317/EGFP cell cultures. (Limon A et al . , (1997), Blood, 90:3316-21 21).
• EGFP expression was analyzed by flow cytometry and bright single cells were sorted on 96-well plates by using an EPICS Elite ESP flow cytometer coupled to an autoclone device (both from Coulter, Miami, FL, USA) .
• Single clones were cultured as previously described. (Limon A et al . , (1997), Blood, 90: 3316-21) .
• the PG13/EGFP7 cell line was subcloned by diluting the cells to 1 cell per well of a 96-well plate. Single subclones were cultured as described and analyzed for transduction efficiency on rhesus BM en UCB CD34+ cells.
• the viral titer of the cell line original and subclones was in the order of 10 5 - 10 6 infectious particles per ml as determined by supernatant titration on cultured human HeLa cells and Rat-2 cells. Absence of replication-competent virus was verified by failure to transfer GFP-expression from a transduced cell population to a secondary population.
• Subcloning of the PG13/SF-EGFP7 virus producer cell line was performed using limiting dilution to grow one cell per well of a 96-well plate culture medium consisting of an enriched version of Dulbecco ' s modified Eagle's medium (DMEM, Gibco, Gaithersburg, MD) .
• DMEM Dulbecco ' s modified Eagle's medium
• Wagemaker G et al . (1980), Cell Tissue Kinet, 13: 505-17 Supplemented with 10% FCS .
• Growing clones were harvested and grown in T75cm 2 flasks until 80% confluency and subsequently tested for transduction efficiency.
• the producer cell lines (PG13/SF-EGFP7 , clone 1, clone 2, clone 3 and clone 5) were grown in T75 cm 2 culture flasks until 80% confluency as described above. Subsequently, 2000 Rat-2 cells and HeLa cells were cultured for 24 hours in dilutions of 0,45 ⁇ m filtered virus supernatant of the different clones of the virus producer cell line m 12 wells of a 24 -wells plate. As a control, 1 well did not contain virus supernatant but culture medium solely. The virus supernatant was removed and substituted with fresh culture medium. The transduced cells were harvested at confluency and the transduction efficiency was determined by flow cytometry (FACSCalibur , Becton &
• the virus titer was determined by calculating the number of cells initially cultured (2000) that were transduced at a certain dilution of the virus supernatant.
• Supernatants containing recombmant retrovirus were generated by culturmg approximately 80% confluent producer cells for 12 hours m culture medium consisting of serum- free enriched version of Dulbecco ' s modified Eagle's medium (DMEM, Gibco, Gaithersburg, MD) .
• DMEM Dulbecco ' s modified Eagle's medium
• DMEM serum-free enriched version of Dulbecco ' s modified Eagle's medium
• Media for all cultures routinely included 100 U/ml of penicillin and 100 ⁇ g/ml of streptomycin.
• the cultures were maintained at 37°C with 10% C0 2 (measured every 15' with read-outs between 9,5% and 10%) m a humidified atmosphere.
• the culture supernatant was subsequently produced and passed through a 0,45 ⁇ m filter.
• Falcon 1008 (35 mm) bacteriological culture dishes were coated with the recombmant fibronectm fragment CH-296 (Takara Shuzo, Otsu, Japan) at a concentration of 10 ⁇ g/cm 2 as described previously. (Moritz T et al . (1996), Blood, 88:855-62).
• CD34- selected UCB, human BM, normal rhBM or rhBM from G-CSF and/or Flt3-L treated monkeys were prestimulated for 2 days in enriched Dulbecco ' s medium with combinations of the human recombmant growth factors Flt3-L (50 ng/ml, kindly provided by A gen, Thousand Oaks, CA, USA) , thromopoietm (huTPO and rhTPO, 10 ng/ml, kindly provided by Genentech, South San Francisco, CA, USA) and stem cell factor (SCF, 100 ng/ml) .
• Flt3-L 50 ng/ml, kindly provided by A gen, Thousand Oaks, CA, USA
• thromopoietm huTPO and rhTPO, 10 ng/ml, kindly provided by Genentech, South San Francisco, CA, USA
• SCF stem cell factor
• the CH-296 fibronectm fragment was premcubated with supernatant containing the pseudotyped vector for 1 hour at 37°C. (Moritz et al . (1996), Blood, 88: 855-62). (Hanenberg H et al. (1996), Nat Med, 2: 876-82). Subsequently, nucleated cells were resuspended in the vector-containing supernatant supplemented with hematopoietic growth factors (HGF) , and added to the dishes. Over a period of 2 days, culture supernatant was replaced completely by resuspendmg nonadherent cells into fresh retrovirus supernatant and HGF. Finally, the cells were harvested and analysed by flow cytometry and cell cycle analysis.
• HGF hematopoietic growth factors
• cryopreserved cells were cryopreserved prior to use, as were the indicated samples of rhesus monkey bone marrow.
• the cells were suspended m HEPES buffered Hanks' balanced salt solution, supplemented with 22,5% foetal calf serum and 7,5% DMSO m a concentration ranging from 20-200 x 10 6 /ml.
• the cells were frozen m ampoules of 1, 1 , 5 or 5 ml volume using a Planer Bio ed Kryo 10 controlled cryopreservation machine during the crystallization at a rate of -1°C per minute.
• cryopreserved cells Prior to use, cryopreserved cells were thawed by the standard so-called "step-wise dilution" method, thoroughly washed and resuspended in the medium used for transduction.
• Rhesus monkey BM (rh BM) and human umbilical cord blood (UCB) cells were titrated from 3xlO ⁇ to 10 3 /ml during the transduction assay.
• the producer cell line was, as standard, cultured in T75 cm 2 flasks filled with 10 ml serumfree medium as described above until 80% confluency. During the transduction, the virus supernatant was refreshed once. After 2 days prestimulation and 2 days of supernatant infection the cells were harvested and the transduction efficiency was determined by flow cytometry.
• NOD/SCID mice Male, specified pathogen-free (SPF) NOD/LtSz-scid/scid mice (NOD/SCID) were bred and housed under SPF conditions and supplied with sterile food and drinking water containing 100 mg/1 ciprofloxac e (Bayer AG, Leverkusen, Germany) ad libitum. Housing, care and all animal experimentation were done m conformity with legal regulations The Netherlands, which include approval by a local ethical committee. All mice received total body irradiation (TBI) at 3.5 Gy, delivered by a 137 Cs source adapted for the irradiation of mice (Gammacell, Atomic Energy of Canada, Ottawa, Canada), 2-4 hours before transplantation.
• TBI total body irradiation
• mice were suspended in 200 ⁇ l H+H and injected I.V. into a lateral tail vein.
• Transplanted cell numbers were 2xl0 5 CD34 + UCB cells.
• the mice were killed by C0 2 inhalation followed by cervical dislocation, both femurs and the spleen were isolated and BM suspensions were prepared by flushing.
• BM cells were analyzed by flow cytometry to determine the percentage human EGFP ⁇ cells m the mouse BM and their multilineage nature determined by flow cytometry. Data were expressed as median (ranged .
• Statistical comparisons were performed according to Mann Whitney U-test. Two tailed P values of ⁇ 0.05, were considered significant. Actual significance levels are indicated in table 1 and in the figures.
• EGFP-transduced CD34+ cells are transplanted into rhesus monkeys subjected to 9 Gy (6 MV X-rays) total body irradiation in cell numbers range from hundred thousand to several millions of CD34+ cells per kg body weight, either immediately following transduction or selected for expression of the EGFP gene by cell sorting using a FACS Vantage cell sorter (Becton Dickinson) .
• the transplantation procedure has been described in detail (Neelis KJ et al . (1997), Exp Hematol , 25:1094-
• the monkeys are followed daily for expression of EGFP in peripheral blood subsets, and weekly for expression in bone marrow subsets, using flow cytometric surface marker labeling to identify the different blood cell lineages.
• the transduction of stem cells using methods of the invention can be reproducibly very high.
• Figure 1 Cell titration of CD34-selected rhesus BM cells (A) and human umbilical cord blood (UCB) cells. (B) during infection with the GALV-pseudotyped PG13/SF-EGFP7 retroviral packaging cell line/vector combination. The highest levels of EGFP + cells were found after transduction of 5xl0/ml rh BM cells or 10 5 /ml UCB cells with 35% and 80%, respectively.
• GM-CFU mu ⁇ ne bone marrow granulocyte/macrophage progenitor cells
• Fibronectm improves transduction of reconstituting hematopoietic stem cells by retroviral vectors : evidence of direct viral binding to chymotryptic carboxy-terminal fragments. Blood, 88: 855-62.

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