the present invention covers the genetic modification of stem / progenitor for the preparation of cells resistant to intrinsic or extrinsic disease such as immune - mediated , inflammatory , viral , bacterial , autoimmune , toxin - mediated disease , aging and / or degenerative diseases . this invention also covers the preparation of cells modified genetically to alter their responsiveness to drug therapy . this patent application covers genetic modification of stem / progenitor or their progeny for the purpose of extending the life of these or other cells and genetic modification of multipotent stem / progenitor and their progeny for treatment of clinical disease , especially in the human nervous system . finally , this patent application covers the transplantation of unmodified stem cells by the same methods . alternatively , stein / progenitor may be modified ( altered gene expression ) through culture techniques to produce a desired cell line , cell type or cell class . such techniques include exposing stem / progenitor to an exogenous agent , such as retinoic acid , or dimethylsulfoxide , promoting differentiation or modification of the stem / progenitor into the desired cell line , such as , for example , a neuronal cell line , but does not exclude the use of physiologic modifiers such as steel factor or other cytokines . there are multiple sources of exogenous stem / progenitor cells . the present invention is directed toward the use of any genetically altered stem cell , progenitor cell , umbilical cord blood stem cell or immortalized cell lines , and / or their progeny for the purpose of treating disease or clinical condition , especially those of the nervous system . however bone marrow stem cells , spermatogonia , and primordial germ cells of the testis are of particular interest and are specifically covered by this invention . a stem / progenitor cell may be induced to differentiate into a desired cell line , cell type , or cell class . in this newly differentiated state the stem / progenitor cell ( and or its progeny ) are considered to be modified . the stem / progenitor cell may also be modified through genetic engineering techniques using dna or rna , encoding protein ( s ) or polypeptide ( s ) promoting differentiation of the stem cell into a specific cell line ( for example , a neuronal cell line , a muscle cell line , or a hematopoietic cell line ), cell type or cell class . the dna or rna may encode a transcription factor found in the particular cell lines , types ( e . g . neurons , glia , muscle ), or classes ( e . g . neural cells , hematopoietic cells , etc .). the term genetic modification refers to alteration of the cellular genotype by introducing natural or synthetic nucleic acids into stein / progenitor or immortalized cell lines and / or their progeny by any means known to the art . alternatively culture conditions that induce permanent changes in gene expression patterns are considered herein to represent genetic modification . modification of stem cells , whether they be derived from the host brain , endogenous donor sources , exogenous donor sources , or cell lines , represents a feasible approach to the treatment of certain human diseases , especially those of the human nervous system . genetic modifications covered by this patent application include , but are not limited to : modifications that alter the activity or amount of metabolic enzymes expressed by endogenous or exogenous stem / progenitor ; modifications which alter the activity , amount , or antigenicity of cellular proteins ; modifications which alter the activity or amount of proteins involved in signal transduction pathways ; modifications which alter the amount or activity of structural proteins ; modifications which alter the amount or activity of membrane associated proteins ( structural or enzymatic ); modifications which alter the activity or amount of proteins involved in dna repair and chromosome maintenance ; modifications which alter the activity or amount of proteins involved in cellular transport ; modifications which alter the activity or amount of enzymes ; modifications which alter the activity or amount of proteins involved in synapse formation and maintenance ; modifications which alter the activity or amount of proteins involved in neurite outgrowth or axon outgrowth and formation ; modifications altering the amount or activity of antioxidant producing enzymes within the cell ; modifications which lead to altered post - translational modification of cellular proteins ; modifications which alter the activity or amount of proteins involved in other aspects of cellular repair , and alterations which increase the lifespan of the cell ( such as production of telomerase ). such proteins as those mentioned above would be encoded for by dna or rna derived from the human genome or other animal , plant , viral , or bacterial genomes . this invention also covers sequences designed de novo . in the first part , this invention relates to the in situ , genetic modification of stem / progenitor cells of the nervous system for the treatment of disease . endogenous stem cells may be modified in situ by direct injection or application of dna or rna vectors , including viruses , retroviruses , liposomes , etc , into the substance of the tissue or into the appropriate portion of the ventricular system . we have modified thousands of stem / progenitor cells and many thousand progeny cells in this manner . our data shows that this manner of modifying progenitor cells results in a tremendous variety of modified cell types throughout the nervous system . we have achieved genetic modification of stem cells in situ in multiple species . although it may be useful to administer neurotrophins ( e . g . brain - derived neurotrophic factor ( bdnf ), basic fibroblast growth factor ( bfgf )) prior to harvesting endogenous cells or at the time of in - situ stem cell modification , it may in most instances not be necessary . either approach is covered by this patent application . the methods of the present invention provide an alternative to pharmacological therapy for the treatment of many diseases . nevertheless , it may be suitable as well for modifying cells to deliver pharmaceuticals beyond the blood - brain barrier for the treatment and alleviation of diseases in the nervous system including psychiatric diseases , or to increase cellular responsivity to pharmacological therapy ( including neoplastic cells ). in the second part , this invention relates to stem and progenitor cells whether they be endogenous cells in situ , or exogenous cells derived from other body regions or even other individual donors . these relatively undifferentiated , self - renewing cells ( herein referred to as stem / progenitor ) are very rare . nevertheless certain sources of stem cells ( such as the spermatogonia and primordial germ cell of the testis ) are accessible and therefore a useful source of replacement cells in the non - fetal human . in vitro genetic modification of exogenous cells or patient &# 39 ; s endogenous cells are performed according to any published or unpublished method known to the art ( e . g . u . s . pat . no . 6 , 432 , 711 , u . s . pat . no . 5 , 593 , 875 , u . s . pat . no . 5 , 783 , 566 , u . s . pat . no . 5 , 928 , 944 , u . s . pat . no . 5 , 910 , 488 , u . s . pat . no . 5 , 824 , 547 , etc .) or by other generally accepted means . successfully transfected cells are identified by selection protocols involving markers such as antibiotic resistance genes . clones from successfully transfected cells , expressing the appropriate exogenous dna at appropriate levels , will be preserved as cell lines by cryopreservation ( utilizing any appropriate method of cryopreservation known to the art ). more particularly , this invention relates to progenitor / stem / spermatogonia cells and / or their progeny ( and any other stem / progenitor cell ) which are modified genetically with dna and / or rna , and or modified through culture techniques whereby such cells become capable of differentiating into a desired primary cell line or cell class , such as neurons , glia , muscle cells etc . throughout this patent application , modified spermatogonia cells and their progeny are corporately referred to as spermatogonia or as modified spermatogonia . it is an object of the present invention to provide modified stem / progenitor ( and any other stem / progenitor cell ) which are capable of differentiating uniformly into a cell line , cell type , or cell class ( e . g . neural cells ), not achievable by previous methods . in accordance with an aspect of the present invention , there is provided a method of producing a desired cell line , cell type , or cell class from stem / progenitor cells . generally , the method comprises culturing spennatogonia under conditions which promote growth of the spermatogonia at an optimal growth rate . the spermatogonia then are cultured under conditions which promote cell growth at a rate which is less than the optimal rate , and in the presence of an agent promoting differentiation of the spermatogonia into the desired cell line , cell type , or cell class ( e . g . neural cells ). a growth rate which is less than the optimal growth rate , is a growth rate from about 10 % to about 90 % ( preferably 20 % to 50 %) of the maximum growth rate for spermatogonia . the growth rates for spermatogonia can be determined from the doubling times of the spermatogonia in one embodiment , when the spermatogonia cells are being cultured under conditions which promote growth of the cells at an optimal growth rate , the spermatogonia are cultured in the presence of a medium including leukemia inhibitory factor ( lif ), and serum selected from the group consisting of : ( i ) horse serum at a concentration of from about 5 % by volume to about 30 % by volume ; and ( ii ) fetal bovine serum at a concentration of from about 15 % by volume to about 30 % by volume . in one embodiment , the serum is horse serum at a concentration of about 10 % by volume . in another embodiment , the serum is fetal bovine serum at a concentration of about 15 % by volume . in yet another embodiment , when the spermatogonia are cultured at an optimal growth rate , the spermatogonia are cultured in the absence of a feeder layer of cells . in one embodiment , the agent ( s ) promoting differentiation of the spermatogonia is / are selected from the group consisting of retinoic acid and nerve growth factor , and the desired cell line , cell type , or cell class is neuronal . in one embodiment , in addition to culturing the cells in the presence of the stimulating agent selected from the group consisting of retinoic acid and nerve growth factor , the spermatogonia are grown in the presence of a cytokine . cytokines which may be employed include , but are not limited to , any of the neurotrophins : nerve growth factor , bdnf , ggf , etc ., bfgf , egf , pdgf , reelin , interleukin - 1 , interleukin - 3 , interleukin - 4 , interleukin - 6 , colony stimulating factors such as m - csf , gm - csf , and csf - 1 , steel factor , and erythropoietin . in a further embodiment , the agent ( s ) promoting differentiation of the spermatogonia is / are selected from the group consisting of dimethylsulfoxide and hexamethylene hisacrylamide , and the desired cell line is a muscle cell line , cell type , or cell class , such as a smooth muscle cell line , or a skeletal muscle cell line , or a cardiac muscle cell line . in one embodiment , the agents is dimethylsulfoxide . in another embodiment , the agent ( s ) is hexamethylene bis - acrylamide . in one embodiment , in addition to culturing the spermatogonia in the presence of agent ( s ) promoting differentiation of the spermatogonia into a muscle cell line , the spermatogonia also are grown in the presence of a cytokine , examples of which are described above . in yet another embodiment , when the spermatogonia are cultured in the presence of the agent ( s ) promoting differentiation of the spermatogonia into a desired cell line , cell type , or cell class , the spermatogonia also are cultured in the presence of fetal bovine serum at a concentration of about 10 % by volume . in a further embodiment , when the spermatogonia are cultured in the presence of the agent ( s ) promoting differentiation of the spermatogonia cells into a desired cell line , cell type , or cell class , the spermatogonia also are cultured on a three dimensional supporting structure . thus , the applicants submit that one may produce a desired cell line , cell type , or cell class from progenitor / stem / spermatogonia cells and / or primordial germ cells of the testis by culturing the progenitor / stem / spermatogonia cells initially under conditions which favor the growth or proliferation of such progenitor / stem / spermatogonia cells at an optimal growth rate , and then culturing the cells under conditions which decrease the growth rate of the cells and promote differentiation of the cells to a desired cell type . in a preferred embodiment , the progenitor / stem / spermatogonia cells cultured in a standard culture medium ( such as , for example , minimal essential medium ), which may include supplements such as , for example , glutamine , and beta .- mercaptoethanol . the medium may also include leukemia inhibitory factor ( lif ), or factors with lif activity , such as , for example , cntf or il - 6 , and horse serum . lif , and factors with lif activity , prevents spontaneous differentiation of the progenitor / stem / spermatogonia cells , and is removed prior to the addition of the agent ( s ). horse serum promotes differentiation of the progenitor / stem / spermatogonia cells into the specific cell type after the addition of the agent ( s ) to the medium . after the cells have been cultured for sufficient time to permit the cells to proliferate to a desired number , the cells are washed free of lif , and then cultured under conditions which provide for cell growth at a decreased growth rate but which also promote differentiation of the cells . subsequently , the cells are cultured in the presence of agent ( s ) promoting differentiation of the progenitor / stem / spermatogonia cells into a desired cell line , cell type , or cell class , and in the presence of fetal bovine serum at a concentration of from about 5 % by volume to about 10 % by volume , preferably at about 10 % by volume . the presence of the fetal bovine serum at a concentration of from about 5 % by volume to about 10 % by volume , and of the agent ( s ), provides for growth or proliferation of the cells at a rate which is less than the optimal rate , while favoring the differentiation of the cells into a homogeneous desired cell type . the desired cell type is dependent upon the agent ( s ) promoting or stimulating the differentiation of the spermatogonia . the spermatogonia also may be cultured on a three dimensional supporting network . for example , the spermatogonia may be placed in a culture vessel to which the cells do not adhere . examples of non - adherent substrates include , but are not limited to , polystyrene and glass . the substrate may be untreated , or may be treated such that a negative charge is imparted to the cell culture surface . in addition , the cells may be plated in methylcellulose in culture media , or in normal culture media in hanging drops . in order to form aggregates in hanging drops of media , cells suspended in media are spotted onto the underside of a lid of a culture dish , and the lid then is placed on the culture vessel . the cells , due to gravity , collect on the undersurface of the drop and form aggregates . in accordance with another aspect of the present invention , there is provided a spermatogonia cell that has been modified with dna or rna encoding protein ( s ) or polypeptide ( s ) which promote ( s ) differentiation of the cell into a specific cell line , cell type , or cell class . the dna or rna encoding protein ( s ) or polypeptide ( s ) promoting differentiation of the spermatogonia cell into a specific cell line , cell type , or cell class is found in the specific differentiated cell line , cell type , or cell class . preferably , the protein or polypeptide which is present in the specific cell line , cell type , or cell class is protein ( s ) or polypeptide ( s ) which generally is not present in other types of cells . in one embodiment , the dna or rna encoding protein ( s ) or polypeptide ( s ) which promote ( s ) differentiation of the spermatogonia cell into a specific differentiated cell line , cell type , or cell class , is present in the desired cell line , cell type , or cell class . in one embodiment , the dna or rna encodes a transcription factor present in neuronal cells , and the specific cell line , cell type , or cell class is a neuronal cell line . in another embodiment , the dna or rna encodes a transcription factor such as the myod gene , present in muscle cells , and the specific cell line is a muscle cell line . in yet another embodiment , the dna or rna encodes a transcription factor present in hematopoietic cells , and the specific cell line is a hematopoietic cell line . in yet another embodiment , the dna or rna encodes a transcription factor dna or rna encodes a transcription factor present in one cell line , cell type , or cell class but the desired cell line , cell type , or class is different from that of the transcription factor . the dna or rna encoding protein ( s ) or polypeptide ( s ) promoting differentiation of the spermatogonia cell into a specific cell line may be isolated in accordance with standard genetic engineering techniques ( for example , by isolating such dna from a cdna library of the specific cell line ) and placing it into an appropriate expression vector , which then is transfected into spermatogonia . appropriate expression vectors are those which may be employed for transfecting dna or rna into eukaryotic cells . such vectors include , but are not limited to , prokaryotic vectors such as , for example , bacterial vectors ; eukaryotic vectors , such as , for example , yeast vectors and fungal vectors ; and viral vectors , such as , but not limited to , lentiviral vectors , adenoviral vectors , adeno - associated viral vectors , and retroviral vectors . examples of retroviral vectors which may be employed include , but are not limited to , those derived from moloney murine leukemia virus , moloney murine sarcoma virus , and rous sarcoma virus , hy , and hiv . plasmid dna containing cdna inserts can be electroporated into spermatogonia . cells are transfected with a plasmid that contains sequences for an antibiotic resistance gene and stable transfectants are isolated based on antibiotic resistance . stable transfected clones are isolated and induced with an appropriate agent , or with leukemia inhibitory factor ( lif ) withdrawal alone , and scored for an increased ability to differentiate in response to these induction signals . clones also are examined to determine if they are differentiating spontaneously in the presence of lif . in accordance with another aspect of the present invention , there is provided a method of producing a desired cell line , cell type , or cell class from spermatogonia . the method comprises engineering spermatogonia with dna which encodes protein ( s ) or polypeptide ( s ) promoting differentiation of the spermatogonia into a specific cell line , type , or class . the spermatogonia then are stimulated with agent ( s ) promoting differentiation of the spermatogonia into the desired cell line , cell type , or cell class . in one embodiment , the dna which encodes protein ( s ) or polypeptide ( s ) promoting differentiation of the spermatogonia into a specific cell line is dna encodes a transcription factor present in neuronal cells and the agent ( s ) is / are selected from the group consisting of retinoic acid and nerve growth factor . alternatively , the cells also may be grown in the presence of a cytokine such as those described above . in another embodiment , the dna which encodes protein ( s ) or polypeptide ( s ) promoting differentiation of the spermatogonia into a specific cell line , cell type , or cell class is dna encodes a transcription factor , such as , for example , the myod gene , present in muscle cells and the agent ( s ) is / are a bipolar agent such as dimethylsulfoxide or hexamethylene bis - acrylamide . alternatively , the spermatogonia also may be grown in the presence of a cytokine . the spermatogonia may be engineered with the dna or rna and cultured under conditions described above . for example , prior to induction , the spermatogonia are engineered with dna which encodes protein ( s ) or polypeptide ( s ) promoting differentiation of the spermatogonia into a specific cell line , cell type , or cell class . then , the spermatogonia may be cultured under conditions which provide for a three - dimensional arrangement of such cells . also , it is to be understood that , within the scope of the present invention , the spermatogonia may be used for gene therapy purposes . the spermatogonia may be engineered with dna encoding a desired therapeutic agent . such engineering may be accomplished by using expression vectors such as those herein above described or others . once the cells are engineered with dna encoding a desired therapeutic agent , the cells then are engineered with dna or rna which encodes protein ( s ) or polypeptide ( s ) promoting differentiation of the spermatogonia into a specific desired cell line , cell type , or cell class , and / or stimulated with agent ( s ) promoting differentiation of the spermatogonia into a desired cell line , cell type , or cell class . the differentiated cells then may be administered to a host , such as a human or non - human host , as part of a gene therapy procedure . the differentiated stem cells may be employed by means known to those skilled in the art to treat a variety of diseases or injuries . for example , stem cells which have differentiated into neuronal cells may be administered to a patient , such as , for example , by transplanting such cells into a patient , to treat diseases such as huntington &# 39 ; s disease , parkinson &# 39 ; s disease , and alzheimer &# 39 ; s disease . such neuronal cells also may be employed to treat spinal cord injuries or chronic pain . also , stem cells which have differentiated into muscle cells may be employed in treating muscular dystrophy , cardiomyopathy , congestive heart failure , and myocardial infarction , for example . the invention will now be described with respect to the following examples ; however , the scope of the present invention is not intended to be limited thereby . undifferentiated progenitor / stem / spermatogonia are maintained in dulbecco &# 39 ; s modified minimal essential medium ( dmem ) supplemented with glutamine , beta .- mercaptoethanol , 10 % ( by volume ) horse serum , and human recombinant leukemia inhibitory factor ( lif ). the lif replaces the need for maintaining progenitor / stem / spermatogonia cells on feeder layers of cells , ( which may also be employed ) and is essential for maintaining progenitor / stem / spermatogonia cells in an undifferentiated state . in order to promote the differentiation of the progenitor / stem / spermatogonia cells into neuronal cells , the progenitor / stem / spermatogonia cells are trypsinized and washed free of lif , and placed in dmem supplemented with 10 % ( by volume ) fetal bovine serum ( fbs ). after resuspension in dmem and 10 % fbs , 1 × 10 6 cells are plated in 5 ml dmem plus 10 % fbs plus 0 . 5 microm retinoic acid in a 60 mm fisher brand bacteriological grade petri dish . in such petri dishes , progenitor / stem / spermatogonia cells cannot adhere to the dish , and instead adhere to each other , thus forming small aggregates of cells . aggregation of cells aids in enabling proper cell differentiation . after two days , aggregates of cells are collected and resuspended in fresh dmem plus 10 % fbs plus about 0 . 5 microm retinoic acid , and replated in petri dishes for an additional two days . aggregates , now induced four days with retinoic acid , are trypsinized to form a single - cell suspension , and plated in medium on poly - d - lysine - coated coated tissue culture grade dishes . the stem cell medium is formulated with kaighn &# 39 ; s modified ham &# 39 ; s f 12 as the basal medium with the following supplements added : 15 microg / ml ascorbic acid , 0 . 25 % ( by volume ) calf serum , 6 . 25 microg / ml insulin , 6 . 25 microg / ml transferrin , 6 . 25 microg / ml selenous acid , 5 . 35 microg / ml linoleic acid , 30 pg / ml thyroxine ( t3 ), 3 . 7 ng / ml hydrocortisone , 1 . 0 ng / ml heparin 10 ng / ml somatostatin , 10 ng / ml gly - his - lys ( liver cell growth factor ), 0 . 1 microg / ml epidermal growth factor ( egf ), 50 microg / ml bovine pituitary extract ( bpe ). this medium will provide for consistent differentiation of the stem cells into neuronal cells , and provides for survival of the neuronal cells for a period of time greater than 3 days , and selectively removes dividing non - neuronal cells from the population ( u . s . pat . no . 6 , 432 , 711 ). the poly - d - lysine promotes the attachment of the neuronal cells to the tissue culture plastic , and prevents detachment of the cells from the dish and the formation of floating aggregates of cells . the cells are cultured for 5 days . upon culturing the cells in the above medium , a culture of cells in which greater than 90 % of the cells are neuronal cells is obtained . such neuronal cells , which express the neurotransmitter gamma amino butyric acid ( gaba ), then may be employed for the treatment of the neural degeneration disease huntington &# 39 ; s disease . through genetic engineering , these cells can be directed to express dopamine ( for the treatment of parkinson &# 39 ; s disease ) or acetylcholine ( for the treatment of alzheimer &# 39 ; s disease ). undifferentiated progenitor / stem / spennatogonia cells are maintained in supplemented dulbecco &# 39 ; s modified minimal essential medium as described in example 1 . the progenitor / stem / spermatogonia cells then are trypsinized and washed free of lif and placed in 1 % ( by volume ) dimethylsulfoxide in dmem plus 10 % horse serum . two days after the addition of dimethylsulfoxide and plating of cells in petri dishes to form aggregates , the aggregates are collected and resuspended in fresh medium plus 1 % dimethylsulfoxide . the aggregates are then plated onto multi - well untreated culture grade dishes without trypsin treatment . one aggregate is plated per well . the aggregates are cultured for 5 days . upon culturing of the cells in multi - well dishes , cell cultures in which greater than 90 % of the aggregates contain contracting muscle cells are obtained . such cells may be used to treat cardiomyopathies , myocardial infarction , congestive heart failure , or muscular dystrophy . progenitor / stem / spermatogonia cells can be isolated using a two - step enzymatic digestion followed by percoll separation . cells can then be resuspended in minimum essential medium ( mem ) supplemented with bovine serum albumin to a final concentration of 10 ( 6 )/ ml . in detail : tubule fragments are accessed surgically and teased apart prior to treatment with 1 mg / ml trypsin , hyaluronidase , and collagenase , and then 1 mg / ml hyaluronidase and collagenase , in mem containing 0 . 10 % sodium bicarbonate , 4 mm l - glutamine , nonessential amino acids , 40 μg / ml gentamycin , 100 iu to 100 μg / ml penicillin - streptomycin , and 15 mm hepes . progenitor / stem / spermatogonia cells are further separated from tubule fragments by centrifugation at 30 × g . after filtration through nylon filters with 77 - and / or 55 - μm pore sizes , cells are collected and loaded onto a discontinuous percoll density gradient . fractions with a purity greater than 40 % progenitor / stem / spermatogonia cells are washed and resuspended to a concentration of cells equivalent to 10 6 progenitor / stem / spermatogonia cells per milliliter . afterwards cells will be cultured and / or stored by any cryopreservation technique known to the art . progenitor / stem / spermatogonia cells can be maintained in media containing 5 ng / ml human recombinant leukemia inhibitory factor instead of on feeder layers . stable transfectants can be isolated , expanded , frozen , and then stored in liquid nitrogen . genetic modification of progenitor / stem / spermatogonia cells may or may not require construction of genetic constructs such as dna or rna vectors . genetic constructs will in most cases consist of a vector backbone , and a transactivator which regulates a promoter operably linked to a heterologous gene nucleic acid sequence . an example of a suitable vector would be a retroviral vector . retroviruses are rna viruses which contain an rna genome . the gag , pol , and env genes are flanked by long terminal repeat ( ltr ) sequences . the 5 ′ and 3 ′ ltr sequences promote transcription and polyadenylation of mrna &# 39 ; s . the retroviral vector provides a regulable transactivating element , an internal ribosome reentry site ( ires ), a selection marker , and a target heterologous gene operated by a regulable promoter . alternatively , multiple genes may under the control of multiple promoters . finally the retroviral vector contains cis - acting sequences necessary for reverse transcription and integration . upon infection , the rna is reverse transcribed to dna which integrates efficiently into the host genome . the recombinant retrovirus of this invention is genetically modified in such a way that some of the retroviral , infectious genes of the native virus have been removed and in certain instanced replaced instead with a target nucleic acid sequence for genetic modification of the cell . the transgene would typically be exogenous dna , in its natural or altered form , from animal or plant species . in many instances the transgene would be altered to contain a signal sequence that directs the transgene &# 39 ; s protein product to be secreted from the cell , possibly for uptake and utilization by adjacent , non - modified , host cells . an example of a method of producing a virus whereby progenitor / stem / spennatogonia cells may be modified is as follows : “ packaging cell lines ” derived from human and / or animal fibroblast cell lines are the result of transfecting or infecting normal cell lines with viral gag , pol , and env structural genes . on the other hand , packaging cell lines produce rna devoid of the psi sequence , so that the viral particles produced from packaging cell do not contain the gag , pol , or env genes . once vector dna containing the psi sequence ( along with the therapeutic gene ) is introduced into the packaging cell , by means of transfection or infection , the packaging cell will produce virions capable of transmitting the therapeutic rna to the final target cell ( e . g . a neuroblast ). the “ infective range ” of this engineered virus is determined by the packaging cell line . a number of amphotrophic packaging cell lines are available for production of virus suitable for infecting a broad range of human cell types . these cell lines are nevertheless generally capable of encapsidating viral vectors derived from viruses which in nature usually infect different animal species . for the example vectors derived from the mmlv can nevertheless be packaged by amphotrophic cell lines . an example protocol for producing a therapeutic viral supernatant will , in general , follow the protocol outlined below : 1 . twenty micrograms of retrovirus vector should be mixed with 2 - 3 micrograms of viral dna containing the selectable marker gene ( e . g . antibiotic resistance gene ) by gentle tapping in 0 . 8 - 1 milliliter of hepes buffered saline ( ph = 7 . 05 ) in a 1 . 5 ml plastic tube . 2 . seventy microliters of 2m ca c12 should be added to the mixture by repeated gentle tapping . 3 . when a blue precipitate first begins to appear within the tube , the product should be gently applied to a 30 % confluent layer of amphotrophic packaging cells ( from any number of commercial vendors ). the dna mixture should be applied only after first removing the medium from the packaging cells . 4 . the packaging cells should be set to incubate for 20 - 30 minutes at room temperature ( 25 degrees celsius ) before transferring them back to an incubator at 36 - 38 degrees celsius for 3 . 5 hours . 5 . add 3 . 5 - 4 milliliters of hepes buffered saline containing 15 % glycerol for 3 minutes then wash cell with dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem )+ 10 % fbs × 2 . 6 . add back dmem + 10 % fbs , and incubate cells for 20 hours at 37 degress celsius . ( excess viral supernatant are immediately stored or concentrated and stored at − 80 degrees celsius ). supernatant may be stored with 5 - 8 micrograms of polybrene which may increase the efficiency of target cell infection . otherwise polybrene may be added just before infection . 8 . stable producer lines are established by splitting packaging cell lines 1 to 20 or 1 to 40 and subsequently incubating these cells for up to 10 days ( changing medium every three days ) in medium containing selective drugs ( e . g . certain antibiotics corresponding to transfected resistance genes ). 9 . after 10 days isolated colonies are picked , grown - up aliquotted and frozen for storage . assay of retrovirus infectivity / titration are achieved by application of a defined volume of viral supernatant to a layer of confluent “ test ” cells such as nih 3t3 cells plated at 20 % confluence . after 2 - 3 cell division times ( 24 - 36 hours for nih 3t3 cells ) colonies of “ test ” cells incubated at 37 degrees in antibiotic - containing medium are counted . the supernatant &# 39 ; s titer are estimated from these colony counts by the following formula : the accuracy of the estimate is increased by testing large volumes of supernatant over many plates of “ test ” cells . application of the therapeutic viral supernatant to target cells may be accomplished by various means appropriate to the clinical situation . transplantation of in vitro modified progenitor / stem / spermatogonia cells may be accomplished in the following manner : under sterile conditions , the uterus and fetuses are visualized by ultrasound or other radiological guidance . alternatively the uterus may be exposed surgically in order to facilitate direct identification of fetal skull landmarks . progenitor / stem / spermatogonia cells can then be introduced by injection ( using an appropriately - sized catheter or needle ) or into the ventricular system , germinal zone ( s ), or into the substance of the nervous system . injections may be performed in certain instances , through the mother &# 39 ; s abdominal wall , the uterine wall and fetal membranes into the fetus . the accuracy of the injection are monitored by direct observation , ultrasound , contrast , or radiological isotope based methods , or by any other means of radiological guidance known to the art . under appropriate sterile conditions , direct identification of fetal skull landmarks are accomplished visually as well as by physical inspection and palpation coupled with stereotaxic and radiologic guidance ( see example 2 ). appropriate amounts of modified progenitor / stem / spermatogonia cells can then be introduced by injection or other means into the ventricular system , germinal zones , or into the substance of the nervous system . the accuracy of the injection will be monitored by direct observation , ultrasound , or other radiological guidance . in certain , neurological diseases , such as huntington &# 39 ; s disease and parkinson &# 39 ; s disease , cells of a specific portion of the brain are selectively affected . in the case of parkinson &# 39 ; s disease , it is the dopaminergic cells of the substantia nigra . in such regionally - specific diseases affecting adults , radiologically - guided transplantation of modified progenitor / stem / spermatogonia cells can be undertaken under sterile conditions . radiologic guidance will include ct and / or mri , and take advantage contrast or isotope based techniques to monitor injected materials . in certain neurologic diseases , such as some metabolic storage disorders , cells are affected across diverse regions of the nervous system , and the greatest benefit will be achieved by introducing modified progenitor / stern / spermatogonia cells ( or immortalized cell lines ) into the tissue in large numbers in a diffuse manner . likewise endogenous cell modification in these disorders would place a premium on modifying a large number of endogenous cells across all affected regions . in the nervous system , these diseases would be best approached by intraventricular injections ( using an appropriately - sized , catheter - like device , or needle ) which would allow diffuse endogenous cell modification or diffuse engraftment of in vitro modified progenitors and cell lines . however , the modified or unmodified stem cells might also be introduced directly into visceral organs , such as the liver , kidney , gut , spleen , adrenal glands , pancreas , and thymus using endoscopic guidance and any appropriately - sized , catheter - like device , allowing specific introduction and infiltration of progenitor / stem / spermatogonia cells cells into the selected organs . the ability of neuronal progenitors to produce mature blood progeny cells suggests that diseases of one organ system may be treated with genetically modified cells from a separate organ system . the treatment of blood disorders ( hereditary spherocytosis , sickle cell anemia , other hemoglobinopathies , etc ,) for instance would involve the injection of modified progenitor / stem / spermatogonia cells directly into the circulation , by large bore intravenous needle or catheter , to “ home ” to the bone marrow , or directly into the bone marrow following surgical exposure of bones and introduction into the marrow space . other diseases might be best approached by injecting modified cells directly into other organs , e . g . liver , gut , spleen , kidney , skin , lungs , etc . the term lesion is non - specific and refers to any area of cell damage or death . modified cells may provide for replacement of neural tissue damaged in various disease such as stroke , trauma , or infection with the exciting prospect that the modified exogenous cells and their progeny would differentiate in a manner appropriate to the host environment . data from animal studies suggests that memory function in mammals may be increased by altered expression of certain neurotransmitter receptors , such as the nmda receptor . thus injection of modified progenitors into the hippocampus and other memory related brain structures may be expected to ameliorate memory loss due to a variety of degenerative disorders . we propose that clinically significant genes might be transferred to modified progenitor / stem / spermatogonia cells and their progeny in an analogous manner for the treatment of neurological and non - neurological disease according to the methods described here . progeny cells modified in this manner demonstrate the exciting prospect of secreting by design or by “ leak ” pathways , gene products other molecules responsible for various clinical diseases . in utero injection of a genetic vector such as a retrovirus , adenovirus , lentivirus , or mmlv - derived retrovirus , for in vivo , in situ , modification of endogenous cells modification of endogenous cells using various genetic constructs , or transplantation of in vitro modified stem / progenitor cells are accomplished by identical surgical procedures . under sterile conditions , the uterus and fetuses are visualized by ultrasound or other radiological guidance . alternatively the uterus may be exposed surgically in order to facilitate direct identification of fetal skull landmarks . concentrated vectors can then be introduced by injection ( using an appropriately - sized catheter or needle ) or into the ventricular system , germinal zone ( s ), or into the substance of the nervous system . injections may be performed in certain instances , through the mother &# 39 ; s abdominal wall , the uterine wall and fetal membranes into the fetus . the accuracy of the injection are monitored by direct observation , ultrasound , contrast , or radiological isotope based methods , or by any other means of radiological guidance known to the art . postnatal injection of a genetic vector such as a retrovirus for in vivo , in situ , modification of endogenous cells modification of endogenous cells or transplantation of in vitro modified stem / progenitor cells ( or immortalized cell lines ) are similarly accomplished in postnatal patients . under appropriate sterile conditions , direct identification of fetal skull landmarks are accomplished visually as well as by physical inspection and palpation coupled with stereotaxic and radiologic guidance ( see example 2 ). appropriate doses of concentrated genetically - modifying vectors can then be introduced by injection or other means into the ventricular system , germinal zones , or into the substance of the nervous system . the accuracy of the injection will be monitored by direct observation , ultrasound , or other radiological guidance . injection of a genetic vector into specific nervous system regions or nuclei in certain , neurological diseases , such as huntington &# 39 ; s disease and parkinson &# 39 ; s disease , cells of a specific portion of the brain are selectively affected . in the case of parkinson &# 39 ; s disease , it is the dopaminergic cells of the substantia nigra . in such regionally - specific diseases affecting adults , radiologically - guided transplantation of genetic vectors into the affected area ( s ) of the nervous system would be undertaken under sterile conditions . radiologic guidance will include ct and / or mri , and take advantage contrast or isotope based techniques to monitor injected materials . delivery of modified or unmodified stem cells by injection in to the circulatory stream in some instances , it may become apparent that stem cells may integrate on their own in sufficient numbers if they are injected into blood stream , either arterial , venous , or hepatic . in time we expect hundreds of diseases and clinical conditions to be treated and / or ameliorated by the present invention . the following represents an incomplete list of example transgenes which we will seek to have expressed by genetically - modified cells , but is no way limiting on the use of this invention : aspartoacylase in the treatment of canavan &# 39 ; s disease ; hexosaminidase a ( subunit alpha ) in the treatment of tay - sach &# 39 ; s disease ; hypoxanthine guanine phosphoribosyltransferase in the treatment of lesch - nyhan syndrome ; huntingtin in the treatment of huntington &# 39 ; s disease ; beta - glucuronidase in the treatment of sly syndrome ; sphingomyelinase in the treatment of type a and type b niemann pick disease ; the b - subunit of hexosaminidase a and hexosaminidase b in the treatment of sandhoffs disease ; alpha - galactosidase a in the treatment of fabry &# 39 ; s disease ; the yet undiscovered mutated gene in the treatment of type c niemann - pick disease ; the glucocerebrosidase gene in the treatment of gaucher &# 39 ; s disease ; the presenilin genes in the treatment of alzheimer &# 39 ; s disease ; the dopamine - related gene in the treatment of parkinson &# 39 ; s disease ; the vi il gene in the treatment of von hippel lindau &# 39 ; s disease . alpha -, beta -, gamma -, and delta - subunits of hemoglobin for the treatment of sickle cell anemia and other thalassemias . these transgenes will generally represent the coding region or portions of the coding region of the normal genes . it is to be understood , however , that the scope of the present invention is not to be limited to the specific embodiments and examples described above . the invention may be practiced other than as particularly described and still be within the scope of the accompanying claims .
we propose here that endogenous stem / progenitor cells of the developing or adult nervous system be genetically modified in situ , to express therapeutically advantageous gene products . furthermore , we propose here that endogenous or other exogenous stem cells or their progeny be genetically modified when appropriate to express advantageous gene products , and that , if exogenously derived , they be transplanted into the ventricular system of the patient nervous system , the germinal zone of the ventricular system , into postmitotic regions of the cns or other organs .