Patent Document

REFERENCE CITED 
       [0001]    U.S. Pat. No. 5,811,301 September 1998 
       OTHER REFERENCES 
       [0002]    Adzick, N. S. M.D., and Longaker, M. T. M.D. (1991) Fetal Wound Healing, Department of Surgery Unversity of California at San Francisco School of Medicine, san Francisco, Calif., Elsevier, N.Y., p-11-120. 
         [0003]    Balinsky, B. I., Dr. Biol. Sc. (1970) Introductin to embryology, 3 rd  ed. W. B. Saunders Company, Philadelphia, Pa. 
         [0004]    Balzarini, J., Hernandez, A. I., Roche, P., Esnouf, R., Karrlson, A., Camarasa, M. J., Perez-Perez, M. J., (2003) Non-nucleoside inhibitors of mitochdrial thymidine kinase (TK-2) differentially inhibit the closely related herpes simplex virus type 1 TK and Drosophila melanogaster multifunctional deoxynucleoside kinase, Molecular pharmacology, vol. 63, num. 2, p-263-270. 
         [0005]    Barrett, J. A., Katayoun Rezvani, Scott Solomon, Anne M. Dickinson, Xiao N. Wang, Gail Stark, Hannah Cullup, Mark Jarvis, Peter G. Middleton and Nelson Chao, (2003) New Developments in Allotransplant Immunology, Hematology, vol. 1, p-350. 
         [0006]    Bone Marrow Tranplants, (1992) Allogenic bone marrow transplants, chapter 4, HLA system, HLA Matching, and HLA typing retrieved Jan. 16, 2005 from bmtnews.org. 
         [0007]    Bornhauser, M. Thiede, C., Platzbecker, U., Jenke, A., Helwig, A. Plettig, R., Frieberg-Ricter, J., Rollig, C., Geissler, G., Lutterbech, K., Oelschlager, U., and Ehninger, G., (2001) Dose-reduced conditioning and allogeneic hematopoietic stem cell transplant from unrelated donors in 42 patients, Clinical cancer research, vol. 7, p-2254-2262, The American Association for Cancer Research. 
         [0008]    Campbell, N. A., Reece, J. B., and Mitchell, L. G. (1999) Biology, Eukaryotic cell cycle, Adison Westley, Longman, Inc., 5 th  ed., p-206-225. 
         [0009]    Ibid. Chase-Griffith transduction of bacterial cells with viral DNA, p-278-280. 
         [0010]    Ibid. Cell cycle, p-109. 
         [0011]    Clark, B. E., and Thein, S. L. (2004) Molecular diagnosis of haemoglobin disorders, review, Clinical and laboratory haematology, vol. 26, issue 3, p-159, retrieved Jan. 30, 2005 from http://www.blackwell-synergy.com. 
         [0012]    Dedov, V. N., Dedova, I. V., Nicholson, G. A., (2004) Equillibrium between cell division and apoptosis in immortal cells as an alternative to the G1 restriction mechanism in mammalian cells, Cell cycle, vol. 3, num. 4, p-491-495. 
         [0013]    Fabry, O. I., Humphries, M. E., Pauliuk, R., Leboulch, P., Hoffman, R. Nag, R El. L., Eaves, C. (2004) expression of an anti-sickling beta globin in human erythroblasts derived from retrovirally transduced primitive normal and sickle cell disease hematopoietic cells, Experimental Hematology, vol. 32, num. 5, p-461-469. 
         [0014]    Fernandez, M. N., Regidor, C. Cabera, R., Garcia-Marco, J. A., Fores, R., Sanjuan, Experimental Hematology, Cordblood forum, (2003) New concepts in cord blood transplantation, vol. 31, p 535-544. 
         [0015]    Gibson, T., and Medawar, P. B. (1943), ( Leukocyte Chimerism linked with Tolerance), Journal of Anatomy, vol. 77, p-299-310. 
         [0016]    Gruenert, D. C., Bruscia, E., Novelli, G., Colosimo, A., Dallapiccola, B., Sangiuolo, F., and Goncz, K., (2003) Sequence-specific modification of genomic DNA by small DNA fragments, Journal of clinical investigation, vol. 112, p-637-641. 
         [0017]    Herzog, E. L., Chai, L., and Krause, D. S., (2003) Plasticity of marrow derived stem cells, Blood, Review in Translational hematology, the American Society of Hematology, vol. 102, num. 10. 
         [0018]    Hosaka, K., Sato, K., Okamoto N., Kazami, A., Sato, K. (2004) Development of reconstituted embryos derived from somaaic cell nuclei in the rabbit, Human cell, vol. 17, num. 1, p-29-32. 
         [0019]    Kohn, D. B., Satake, N. (2001) Immune responses and tolerance in gene therapy, Division of Research Immunology/B.M.T., he John Connell Gene Therapy Program, Children&#39;s hospital Los Angeles, Calif., University Southern California, Keck School of Medicine. 
         [0020]    Li, Q., Peterson, K. R. Fang, X., Stammatoyannopoulos, G., (2002) Locus Control Region, Blood, vol. 100, p-3077-86. 
         [0021]    Lefrere, F., Levy, V., Makke, J., Audat, F., Cavazzana-Calvo, M., Miclea, J., m. (2004) Successful peripheral blood stem cell harvesting with grandulocyte colonu-stimulating factor alone after previous mobilization failure, Hematologica, vol. 89, num. 12, P-1532-1534. 
         [0022]    Mansour, et. Al. (1988) Neomycin resistant gene as a selection process, Nature, London, vol. 336, p-348. 
         [0023]    Nienhuis, A. W., (1978) Hemoglobin switching new experimental model, Journal of Laborataoary and clinical Medicine, vol. 91, num. 6, -857-861. 
         [0024]    Oh, I. H., Fabry, M. F., Humphries, R. K., Pawliuk, R., Leboulch, P., Hofman, R., Nagel, R. I., and Eaves C. (2004) Expression of an anti-sickle beta-globin in human erythroblasts derived from retrovirally transduced primitive normal and sickle disease Hematopoietic cells, Experimental Hematology, vol. 32, num. 5, p-461-469. 
         [0025]    Roitt, Ivan, Brostoff, Johnathan and Male, David, (1985) Immunology, “Tolerance and Auto-immunity”, Chapters 12 and 23, Gower Medical Publishing, London, New, York. 
         [0026]    Rood, P. M., Calafat, J., Von Dem Borne, A. E., Gerritsen, W. R., Van der schoot, C. E., (2000) Immortilization of bone marrow endothelial cells: Characterization of new cell line, European journal of clinical investigation, vol. 30, Number 7, p-618-629. 
         [0027]    Rund, D., Dobkin, C., and Bamk, A., (1987) Regulated expression of amplified human beta-globin genes, Blood, vol. 70, num. 3, p-733-739. 
         [0028]    Savitt, T. L., and Goldberg, M. F. (1989) -Herrrick&#39;s 1910 case report of sickle cell anemia, The rest of the story, The Journal of the American Medical Association, vol. 261, num. 2, p-266-271. 
         [0029]    Scheuermann, M. O., Tajbakhsh, J., Kurz, A., Saracoglu, K., Eils, R., Lichter, P., (2004) Topology of genes and non-transcribed sequences in human interphase nuclei, Experimental cell biology, vol. 310, num. 2, p-266-279. 
         [0030]    Schroeder, W. A. Ph. D., Jakway, J., Powars, D. M.D., (1972) Detection of hemoglobin S and C at birth: a rapid screening procedure by column chromatography, California Institute of Technology, Pasadena Calif. 
         [0031]    Services and Resources provided (n.d.) The cell and gene bank retrieved Jan. 26, 2005 from 
         [0000]    http: www.ihwg.org/shared/cbankover.htm 
         [0032]    Shinar, D., Yoffe, O., Shani, N., and Yaffe, D. (1989)Regulated expression of muscle-specific genes introduced into mouse embryonic stem cells: inverse correlation with DNA Methylation, Differentiation, vol. 41, num. 2, p-116-126. 
         [0033]    Stevens, A., Wang, Y., Bremer, K., Zhang, J., Hoepfner, R., Antonious, M., Schoenberg, D. R., and Maquat, L. E., (2002) Beta-globin mRNA decay in erythroid cells: UG site preferred endonucleolytic cleavage that is augmented by a premature termination condon, Proceedings of the National Academy of Science, vol. 99, issue 20, p-12741-12746. 
         [0034]    Steward, S., (1992) The Bone Marrow Transplants, The Transplant, and the engraftment, chapter one, p-15. BMT Newsletter, HIGHLANDPARK, Ill. 
         [0035]    Tuan, D., Biro, P. A., deRiel, J. K., Lazarus, H., and Forget, B. G., (1979) Restriction endonuclease mapping of the human gamma globin gene loci,  Nucleic acids Research , vol. 6, num. 7, p-2519-2544. 
         [0036]    United States Patent and Trademark Office. (November, 2006). PTO-EBT files. Retrived Nov. 2,2006 m from http://www.uspto.gov/. 
         [0037]    Van Pelt, K., De Haan, G., Vellenga, F., and Daenen, S, M. (2005) Administration of low dose cytarabine result in immediate S-phase arrest and subsequent activation of cell cycling in murine stem cells, Experimental Hematology, vol. 33, num. 2, p-226-231. 
         [0038]    Weaver, S., Haigwood, N. L., Hutchinson, C. A., III and Edgell, M. H. (1979) DNA Fragment of the Mus Musculus beta-globin haplotypes Hbbs, Proceedings of the National Academy of Science, USA, vol. 76, p-1385-1389. 
         [0039]    Williams, B. M. (Mar. 28, 1956-2005) Unpublished personal research and theorectical papers, file (BMWI Research and Development). 
         [0040]    Zhou,T. Kim, J., Kalani, A. F., Kim, K., Dunn, W., Jo., Nepomuceno, E., and Liu, F., (2002) In vitro selection of external guide sequences for directing RNase P-Mediated inhibition of viral gene expression, Journal of Biological Chemistry, vol. 277 num. 33, p-112-120. 
       DESCRIPTION OF THE INVENTION 
       [0041]    In 1956 the inventor sought out to cure sickle cell anemia when it was not heard of in the medical community. The discovery came when viewing a blood cell developmental chart. This was 1970s. When these cells (Stem cells) were found to take on the characteristics of the environment, it was the inventor&#39;s ideas that this might help with the GvHD of 1970. Later the inventor realized he had stumbled upon a way to replace all the tiny blood cells in the body. Much research had to happen that the inventor was not allowed to take part in. In the 1980s stem cells were being transplanted into humans and it was met with some success. Although the GvHD and HvGD still proved to be problematic. One of the problems was whether the hemoglobin F gene was being activated from without the cell by a factor since control of Hbg F synthesis could help the condition of Sickle cell. It was found that that there is a factor that regulates the switching of fetal and beta hemoglobin. This factor is a protein which is not the same in not the same in all individuals. Inventor investigated( entire nuclear insertion 1982-1986, and studied transfection from 1973-1982) (Williams, 1990) which would alter the Hbg SS to Hbg AA or Gamma, but it would also alter the HLA. This invention finds a way around this problem by transecting recipient&#39;s stem cells with Hbg AA or gamma genes, thereby sparing the HLA type to be perfectly as the original stem cell line. Doctors can decide if the patients need full cell replacement or just globin protein replacement on a case by case basis. Doctors can also decide if they desire multi-potent stem cell transfection or insertion; or progenitor transfection or insertion. In a full cell replacement the HLA type will need to be matched. A method by which Hbg SS can be replaced and the donor HLA remains Intact across the continuum of solid elements of the blood and molecular species such Il-1, GM-CSF, and ICAM. 
       SUMMARY OF THE INVENTION 
       [0042]    The invention is an in vivo procedure for curing Sickle cell anemia and other disease defined as cellular dyscrasias. The procedure avoids GvHD and HvGD by either altering the defective protein by progenitor transfections for uni-directional differentiation, (i.e. red cells vs. lymphocytes). When the condition calls for change of a protein across the solid elements the transfections will be in the Hematopoietic multi-potent stem cells with renewal capability. The transfections gene therapy is site specific by using cDNA probes, restriction enzymes, transfection or insertion, chaperones, and cell cycling specificity. The transfections are general when the desired gene is introduced in the nuclear domain without site specificity. The transfected cells are cultured and prepared for transplantation. Hematopoietic multi-potent stem cells produce all solid elements, and progenitors produce a specific solid element defined by the specific progenitors. A universal donor is selected for HLA transfections. The recipient&#39;s own cells are chosen for other protein gene therapy replacement therapy, such as HbgSS. clinically appropriate amounts of HbgSS will be replaced by HbgAA and or HbgF to increase oxygen saturation by the recipient&#39;s red blood cells. Thymus will accept recipient&#39;s own lymphocytes thereby preventing GvHD and HvGD. 
         [0043]    It is the object of this invention to replace HbgSS in sickle cell anemia patient&#39;s blood cells, and or replace a clinically appropriate amount of progenitor cells to replace therapeutic amount of HbgSS laden cells with HLA compatible HbgAA or F producing Hematopoietic progenitor cells. 
         [0044]    Sigma factor is the glycoprotein factor that when it binds it&#39;s receptors the end stage protein is altered in amino acid sequence hence from HbgF to HbgAA. The switch is at the mRNA level splicing to production of various mRNAs. As many as three Hbgs have been found in a single red blood cell. The hemoglobin producing program switches from producing HbgF to producing HbgAA (Beta-hemoglobin). 
         [0045]    The invention consist of a method to replace the beta globin gene in renewable multipotent stem cells at specific sites in the genome to cure sickle cell anemia such that the globin vector only express it self into solid elements from the Hematopoietic progenitors, or any other progenitors for other conditions. Transfection into the recipient&#39;s stem cells would avoid the GvHD and HvGD situation. Beta globin gene chaperones lead HbgAA or HbgF to restricted site in genome chromosome 11. 
         [0046]    Stem cells are arrested in interphase and nuclei removed, for HbgAA binding and restriction, using HbgAA cDNA probe. HbgAA DNA with valine in the number 6 position of the hemoglobin chain will be removed, and the HbgAA with glutamate in the number 6 position of the hemoglobin chain, or the HbgF will be annealed to the genome and the nucleus reconstituted and replaced back into recipient&#39;s stem cell cytoplasm and nuclear envelope. 
         [0000]    It is the objective of this invention to restrict the globin gene stem cell interphase. By introducing the Sequence Listing into the stem cell the thymus will deplete those lymphocytes that are not tolerant to the recipient, transplants performed before age 1 or 2 years of age will dimenish an immune attack, and reduce restraint of autologous transplants thereby allowing iso and allo-type transplants, after age 2 only autologous and type O transplants are indicated. Diagnosed the condition, identify missing protein, for each clinical condition. Use triflourinated nucleotides (anti-metabolites) only when live un-attenuated viruses are used in the transfection process. Various viral vectors are available with different capabilities. 
       The natural beta hemoglobin sequence is 5′-ε-γg-γa-σ-β-3′. 
     DESCRIPTION OF THE BACKGROUND ART 
       [0047]    In 1910 James B. Herrick presented an article on a case of anemia with sickle cells. 
       In 1943 Medawar and associates discovered the link, or lack thereof, between immature cells and Tolerance. 
       [0048]    In the 1950s to cure a genetic blood disease was mire childhood dreams and unheard of. Scientist learns to diagnose sickle cell anemia using cellulose acetate chromatography and citrate agar chromatography. Early embryologist suspected stem cells to be usable to treat human diseases in 1954 but there were no specifics to a cure of any one particular disease or disease type. In 1956 the inventor thought to cure Sickle cell anemia. Stem cells were adopted because stem cell or embryonic cells have the ability to take on the morphological characteristics of the surrounding tissues when transplanted in the same embryo or different species, and they produce all solid elements. Stem cell transplants of the 1980s met with some success however, there still remained the GvHD and HvGD problem. This concept was clarified by the inventor and taken several steps further to replace large numbers of red blood cells while avoiding the HLA problem of GvHD and HvGD. The avoidance of the HLA problem was not tolerated as the immature cells engrafted into the recipient&#39;s bone marrow stroma as hoped. This avoidance of the HLA problem had to be overcome by additional means. Viral transfections were discovered by Chase and his associate and other scientist began introducing genes into cells for various purposes, such as to produce hormones. Transfections of progenitor hematological cells left the door open for Hematopoietic stem cell transfections and gene insertion or gene therapy. The ability to replace red blood cells that are defective in a protein or group of proteins, avoid the GvHD and HvGD, plus replace the defective protein such as HbgSS with varying amounts of HbgAA or HbgF has still proven difficult if not impossible. Such an approach has been invented, and is described below.
 
The ability of stem cells to take on the character of the surrounding is limited by it&#39;s inability to return to embryonic stem cell from peripheral stem cells, however stem cells from the periphery can function as Hematopoietic stem cells to produce progenitors of all solid elements in the bone marrow or the embryo. The source of stem cells can be from the embryo, bone marrow, or peripheral blood stream. Any of these stems cells can function in the other environment.
 
       FIELD OF THE INVENTION 
       [0049]    This invention relates specifically to curing Hematopoietic Blood diseases by transplanting clinically useful quantities of perfectly matched stem cells (that differentiate into mature Hematopoietic Pluripotent cells) into autologous host. 
         [0050]    In 1956 the inventor sought out to cure sickle cell anemia when it was not heard of in the medical community. The discovery came when viewing a blood cell developmental chart. This was 1970s. When these cells (Stem cells) were found to take on the characteristics of the environment, it was the inventor&#39;s ideas that this might help with the GvHD of 1970. Later the inventor realized he had stumbled upon a way to replace all the tiny blood cells in the body. Much research had to happen that the inventor was not allowed to take part in. In the 1980s stem cells were being transplanted into humans and it was met with some success. Although the GvHD and HvGD still proved to be problematic. One of the problems was whether the hemoglobin F gene was being activated from without the cell by a factor since control of Hbg F synthesis could help the condition of Sickle cell. It was found that that there is a factor that regulates the switching of fetal and beta hemoglobin. This factor is a protein which is not the same in not the same in all individuals. Inventor investigated( entire nuclear insertion 1982-1986, and studied transfection from 1973-1982) (Williams, 1990) which would alter the Hbg SS to Hbg AA or Gamma, but it would also alter the HLA. This invention finds a way around this problem by transecting recipient&#39;s stem cells with Hbg AA or gamma genes, thereby sparing the HLA type to be perfectly as the original stem cell line. Doctors can decide if the patients need full cell replacement or just globin protein replacement on a case by case basis. Doctors can also decide if they desire multi-potent stem cell transfection or insertion; or progenitor transfection or insertion. In a full cell replacement the HLA type will need to be matched. A method by which Hbg SS can be replaced and the donor HLA remains Intact across the continuum of solid elements of the blood and molecular species such Il-1, GM-CSF, and ICAM. 
     
     
       DESCRIPTION OF THE FIGURES 
         [0051]      FIG. 1.0  Peripheral human blood smear. 
           [0052]      FIG. 2.0  Scanning electron micrograph of sickled and normal red blood cells. 
           [0053]      FIG. 3.0  Three dimensional hemoglobin beta amino acid chain. 
           [0054]      FIG. 4.0  Quaternary structure of HbgAA with two alpha chains, and two beta chains. 
           [0055]      FIG. 5.0  Major histo-compatibility and viral transfection scheme. 
           [0056]      FIG. 6.0  Blood chart showing differentiation of all solid elements from multi-potent stem cells and progenitors. 
           [0057]      FIG. 7.0  The phases of the cell cycle. 
           [0058]      FIG. 8.0  LCR region of beta like globin gene. 
           [0059]      FIG. 9.0  Human stem cell cycle, and arrest for gene transfer. 
           [0060]      FIG. 10  Hemoglobin Alu gene family sequences. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0061]    The expression from type O blood is preferred because of a lack of other blood type antigens, for patients with or without functional thymus ( The transplanted cells will replace all solid elements of the blood, of just the element desired) Transplants are for all patients. The multi-potent stem cells will produce all solid elements in physiological amounts. Type O cells will be devoid of other blood group antigens, except where the recipient&#39;s cells are used for the transplant. Progenitor cells may be of any of the Hematopoietic series: Colony forming unit-granulocytes, erythrocyte, monocytes and megakaryocyte, eosinophils 
         [0000]    The bone marrow produces all solid elements and their progenitors. These cells respond to a number of regulators that promote maturation, and differentiation, protein switching such as colony stimulating factor for each progenitor, stem cell growth factor, and sigma factor. 
         [0062]    As each protein system is different, it will call for different measures to meet the demands of the particular patient. Hbg BETA has two chains of alpha hemoglobin and two chains of beta hemoglobin. See appendix  FIG. 4.0 . 
         [0063]    The expression of progenitors for all solid elements with specific differentiated end products such as hemoglobin being corrected from HbgSS to HbgAA and or HbgF. This done with or without altering the HLA genes. To produce clinically appropriate amount of red blood cells with HbgAA and or HbgF to increase the oxygen carry capacity of the recipient&#39;s red blood cells, there by curing the clinical problem of sickle cell crisis. 
         [0064]    For the purpose of this specification and the claims the following definitions and abbreviations will be used: 
         [0065]    1. Hemoglobin (Hbg) -oxygen carrying protein of the red blood cell of humans. 
         [0066]    2. Fetal Hemoglobin- HbgF, carry oxygen with a greater affinity. 
         [0067]    3. Hemoglobin beta (HbgAA)- normal oxygen carrying protein which is defective in sickle cell anemia. 
         [0068]    4. Multi-potent stem cell-stem cell capable of expressing all solid element progenitors. 
         [0069]    5. Solid elements- erythrocytes or red blood cells (RBC), lymphocyte which cause GvHD and HvGD, neutrophiles which are involved in inflammation, megakaryocyte which are precursors of platelets, monocytes the precursors of macrophages, and basophile. 
         [0070]    6. Graph versus host disease- GvHD the recipient of a transplant is not tolerant of the transplant and the transplant began to attack the host or recipient as foreign. Carried out by donor lymphocytes. 
         [0071]    7. Host versus graph disease- HvGD the host immune cells are not tolerant with the transplant and the host cells immune cells began to attack the transplant or graph. Carried out by recipient&#39;s lymphocytes. 
         [0072]    8. Progenitor- Cells derived from multi-potent stem cells that are capable of expressing and producing each of the solid elements specifically as they differentiate. 
         [0073]    9. Universal donor cell- used only when HLA has to be altered, formally known as “type O” blood which does not carry lansteiner antigen such as (i.e. A antigens, B-antigens, and therefore does not cause the production of the alternate antibody (A produce anti-B, and B produce anti-A antibodies which are responsible for the formation of antigen antibody complexes that are harmful to the human blood system. 
         [0074]    10. Bone marrow- medulary site for all blood solid element formation. 
         [0075]    11. Differentiation and switching- process by which immature red cells and other solid elements become the producer of the final type of protein programmed by ontogeny. (e.g. HbgF cells becoming HbgAA cells). The immature cells becomes a mature cell. 
         [0076]    12. Cell cycle- the growing cell goes through a cell cycle from Mitosis to interphase and back to mitosis. Interphase consist of Go, G1, S, and G2 phases. 
         [0077]    The invention is based on the fact that in order to produce HbgF and or HbgAA in a sickle cell anemia individual continually the multi-potential stem cells must be altered at the beta globin gene such that only the erythropoietic progenitors produce the final product which are red blood cells with multiple Hbgs such as HbgAA and or HbgF. This method does not have to alter the HLA antigens and therefore produce perfectly matched and compatible cells that do not produce GvHD or HvGD. The progenitors under the influence of the recipient&#39;s bone marrow environment will regulate the differentiation process. 
       Typical Protocols are Described Below Stem Cell Preparation: 
       [0078]    As a method that follows the trend, heparinized bone morrow or peripheral stem cells by aspiration from the ileac crest, or venipuncture of the brachial vein of the arm respectively of the recipient or donor. O type blood is preferred where the HLA has to be altered. Accepted surgical and venipuncture procedures are used to collect the above samples of stem cells. One unit or 500 ml of blood is stored at −170 degrees Fahrenheit for later use. Reticulum is removed from the bone marrow by passing the marrow through a micro nylon mesh. The stem of peripheral blood are siphoned from the top of the blood sample after centrifugation. 
       Culture Medium 
       [0079]    Bone marrow and peripheral stem cells are transferred to a long term medium consisting of AIM-V medium (Gibco, Grand Island, N.Y.) supplemented with insulin (Eli lilly &amp; Co. Indianapolis, Ind.) at 10 ml mu. g/ml, human albumin (American Red Cross, Washington, D.C.) 50 mg/ml, saturated human ferritin (Sigma Co., St Louis, Mo.) at 200 mu.g/ml, hydrocortisone (sodium succinate) (Upjohn Co. Kalamazoo, Mich.) at 10 sup.-6 M, cholesterol (C3045, Sigma Chemical Co.) at 7.5 mu. g/ml, and liposyn II (10% Abbot Labs., No. Chicago, Ill.) at 0.05 l.ml medium. In addition, penicillin G potassium (Roerig div of Pfizer, Inc. New York, N.Y.), gentamicin sulfate (Schering Corp., Kenilworth, N.J.) and amphotricin B (Bristol Myers-Squibb, Princeton, N.J.) are added to the stem cells as preservatives. Iscove&#39;s modified Dulbecco, Fisher&#39;s or Eagle&#39;s media are used. In addition, fetal calf serum or horse serum may be substituted for human serum. 
       Arresting Cells in Interphase 
       [0080]    The stem cells are arrested at interphase by decreasing the oxygen concentration after Van Pelt (2005). Treat stem cells with Ara-C (100 mg/kg), using 7-aminoactinmycin-D (7-AAD) for DNA staining. Treat with bromodeoxyuridine, 2 hrs later cells cease to incorporate BrU, after 4 hrs s-phase arrested cells began to activate. 28% of cell will incorporate BrU at 20 hrs. After 72 hrs the cells recover from the arrest, returning cells to Go phase. 
       Transfection: 
       [0081]    Removal of the interphase nucleus, excision of the defective globin gene and replacement of beta globin gene with HbGAA or HbgF, annealing of excised DNA and replacement of nucleus. Cells are infected with the virus-A phophoglycerate kinase promoter driven expression of a green fluorescence protein (GFP) cDNA, and an anti-sickle HbgAA or HbgF globin genes under the control of HS2, HS3, HS4 enhancers. 
       Bioreactors 
       [0082]    Transfection of stem cells and isolate them to be tested for desired progenitor incorporating the HbgAA or HbgF genes. And Bioreactor culture systems, to mutiply the stem cells. Opticell. T.M. Optocore.TM. ceramic core S-51, S451 (flat surface area 23.8.sup.2), S-1251 (flat surface area 10.4m.sup.2) or S-7251 (Cellex Biosciences, In., Minneapolis, Minn.) are preferred. These bioreactors are initially sterile perfused, preferably for 1-3 days, with sterile deionized water to remove any toxic substances adhering to the core. Therefore, the core is perfused for a brief period (less than 24 hrs.) with sterile 25% (w/v) human serum albumin in order to coat the core with protein. The bioreactor core is next perfused for 4-24 hrs with a sterile solution of an anticoagulant, preferably heparin sulfate, 100 U/mL (Upjohn Co.) as a source of glycosaminoglycan and to prevent cell clumping during stem cell inoculation. Following this preparation, the core is conditioned by perfusing it with sterile human stem cell medium (see culture mediums above), preferably for about 24 hours, prior to inoculating the bioreactor with stem cells. The stem cells that produce the altered Hbg will be cultured in a bioreactor (as above) to clinically useful number of stem cells for transplant. For procedures see (Oh, 2004). Self-renewal cells take up the viral transfected beta globin gene. 
       Bioreactor Culture System 
       [0083]    The culture system consist of a variable number of bioreactors connected to the medium source by sterile plastic tubing. The medium is circulated through the bioreactor with the aid of a roller or centrifugal pump (e.g., KOBE&gt;TM). Probes to measure pH, temperature, and O.sub.2 tension are located in line at points immediately before and following the bioreactor(s). Information from these sensors is monitored electronically. In addition, provision is made for obtaining serial samples of the growth medium in order to monitor glucose, electrolytes, Growth factors, and other nutrient concentrations. 
       Inoculation of Bioreactor with Altered Pluripotent 
     Hematopoietic Stem Cells 
       [0084]    Multiplying, and Altered Pluripotent Hematopoietic Stem cells 
         [0085]    Appropriate for the size of the bioreactor, at a concentration of about 2 times. 10sup.7 cells/ml., are mixed with an equal volume of autologous fresh stem cells are injected into the bioreactor. Circulation of growth medium is interrupted for a period of about 1-4 hours such that the cells are allowed to attach to the core of the bioreactor core capillaries. Thereafter, the circulator pump is engaged and the growth medium pumped through the system at an initial rate determined by the size of the reactor, atypical rate is about 24 ml/min. Gas exchange occurs via silicone tubes (surface area=0.5 m.sup.2) within a stainedless steel shell, or by a conventional membrane oxygenator. O.sub.2 and pH are monitored continuously by polarographic O.sub.2 probes and autoclavable pH electrodes, respectively. Flow rates are adjusted so as to maintain an optimal O.sub.2 tension (a partial pressure of at least about 30-50 mm of Hg) and optimal pH (7.30-7.45). 
         [0086]    When an appropriate number of vector carrying stem cells have been obtained (approximately 5-10 mililiters), as determined by oxygen utilization of the system, a second bioreactor may be connected to the system, and cells fed directly into this second bioreactor. Thereafter, the second bioreactor is flushed with growth medium containing a high concentration (e.g. 10,000 U/mL) of EPO or other differentiation factors, and maintained for 1-3 days for final maturation of the desired blood components, (i.e. multi-potent-potent stem cells). 
       Cell Harvest and Processing 
       [0087]    The bioreactor(s) is (are) mated with a conventional cell separator, and the cells are collected from the core or capillaries with gentle agitation. Harvested blood cells are processed in an automated cell separator and placed in sterile blood bags for transfusion. 
         [0088]    Bags of stem cells may be irradiated conventionally and tested for any contamination during refrigeration for 1-3 days. Neomycin resistant gene as a selection process, after (Mansour, 1988). Efficiency may be as high as 85%. 
         [0089]    Thus, the invention can also provide a single multipotent stem cell line species for the cure of various previously uncurable diseases. This achieved by expanding the culture of multi-potent Hbg globin altered self-renewing Hematopoietic stem cells in the bioreactor until cell numbers are clinically appropriate to re-introduce them back into the patient or the recipient.

Technology Category: 8