CA2177998A1 - In utero gene therapy for fetuses - Google Patents

Abstract

A process for effecting gene therapy in utero in a fetus, which comprises transducing fetal cells in vivo with at least one nucleic acid sequence encoding a therapeutic agent. The fetal cells may be transduced with a viral vector (such as a retroviral vector) which includes the nucleic acid sequence encoding the therapeutic agent. The viral vector may be contained in a viral supernatant which is administered to the fetus, or may be generated by a producer cell line which is administered to the fetus.

Description

~ WO95115167 2~ 77998 PCT/US94"38ll IN u~rFRn ~ THERAP~ FOR FETUS~S
This invention relates to gene therapy, and in particular to n utero gene therapy. More particularly, this invention relates to ' vivo gene therapy for fetuses by administering to a fetus at least one nucleic acid sequence ~ncofling a therapeutic agent.
Certain inherited metabolic diseases, such as Hurler ' s syndrome, J,esch-Nyhan disease, Tay Sachs disease, and alpha-thalassemia, may produce irreversible damage to the fetus before birth. Infants born with other inherited diseases such as, for example, adenosine tll-A~;nA~e (ADA) deficiency, appear normal at birth; however, such disease6 are manifested shortly thereafter. The above disorders, whether manifested before or after birth, could be treated if gene therapy could be accomplished safely n utero.
Rantoff, et al., Blood, Vol. 73, No. 4, pgs. 1066-1073 ~March 1989 ), discloses the retroviral-mediated transfer of a neomycin resistance (neoR) gene into fetal sheep hematopoietic cells by PYrhAn~e transfusion. In such a procedure, blood was obtained from a sheep fetus, and mononuclear cells were harvested. The cells then were transduced eY. vivo with retroviral vectors including the neoR gene or a cDNA for human adenosine d~Am; nA~e (ADA) .
The transduced cells then were reinfused into the sheep fetuses. After birth, the lambs were PyATn;n~d for presence -Wo 95/15167 21~ 7 ~ ~ ~ PCT/US94113811 of a functioning neo~ gene. ~. Out of ten lambs analyzed, six were positive for G418 resistant hematopoietic-progenitor cells. One sheep had blood cells which expressed the neo~
gene for more than two years after birth.
Such an ~rrhiqn5e transfusion procedure, however, requires that one needs to wait until a period of time late in gestation in order for the fetus to be of a sufficient size in order to obtain sufficient blood cells to effect the gene transfer, and certain genetic diseases could be treated more successfully if gene transfer into fetal cells could be ef f ected early in gestation . In addition, such a procedure requires multiple T~-nirlllAtions of the fetus, which increases the risk of damage to the fetus. Also, transduction takes place only in cells removed from the f etus .
It therefore i6 an object to the present invention to provide gene therapy for a fetus wherein genes encoding therapeutic agents may be tran6duced into fetal cells early in gestation, and to provide i ~Iv~d transduction of 6tem cells of the fetus.
In accordance with an aspect of the present invention, there is provided a process for effecting gene therapy in ViVQ in a fetus. The process comprises transducing fetal cells n v vo with at least one nucleic acid (DNA or RNA) sequence encoding a therapeutic agent.
The term "nucleic acid sequence" as used herein, means a DNA or RNA molecule, and includes complete and partial gene sequences, and includes polynucleotides as well. Such term also includes a linear series o~ deoxyribonucleotides or ribonucleotides connected one to the other by pho6phodiester bondE between the 3 ' and 5' carbons of the ad j a cent pento s e s .
The term "therapeutic" as used herein is used in a generic sense and includes treating agents, prophylactic agents, and replacement agents.

~ WO 95/15167 217 7 9 ~ ~ PCTIUS94113811 The process of the pre6ent invention may be carried out during any stage of gestation, including the yolk sac stage. Such process may be applied to humans, wherein a human fetus may be injected intraperitoneally with a needle which is guided into the fetus by ultrasound, in a manner 6imilar to that in which a fetus is given a blood transfusion for the treatment of alpha-thalessemia.
The nucleic acid sequence which encodes the therapeutic agent is contained in an appropri2lte expression vehicle which tr~nc~ rpc the fetal cells. Such expression vectors include, but are not limited to, eukaryotic vectors, prokaryotic vectors (such as, for example, bacterial vectors ), and viral vectors .
In one embodiment, the expression vector is a viral vector. Viral vectors which may be employed include, but are not limited to, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and Herpes virus vectors.
Preferably, the viral vector is a retroviral vector.
In a preferred ` -~ir t, a packaging cell line is transduced with a viral vector containing the nucleic acid sequence Pnro~1inr~ the therapeutic agent to form a producer cell line which includes the viral vector. The producer cells then are administered in vivo to the fetus, whereby the producer cells generate viral particles capable of transducing fetal cells. Such fetal cells may be located throughout the body and include, but are not limited to, somatic and gPrTn;nAl cells, including bone marrow cells, including hematopoietic stem cells; peripheral blood cells;
cells of the central nervous system, including brain cells;
lung cells; kidney cells; testicular cells; ovarian cells;
~nd liver cells.
In a pref erred ~ _ ' i L, the viral vector is a retroviral vector. Examples of retroviral vectors which may be employed include, but are not limited to, ~oloney DIurine T.Pl~k~oi A Virus, spleen necrosis virus, and vectors 2 ~ 7 ~ ~ ~ 8 PCrNS94/13811 derived from retroviruses such as- Rous Sarcoma Virus, Harvey Sarcoma Virus, avia~i leukosis virus, human nri~f;t i~n~y virus, myeloproliferative sarcoma virus, and mammary tumor virus. Preferably, the retroviral vector is an infectious but non-replication competent retrovirus.
However, replication competent retroviruses may also be used .
Retroviral vectors are usef ul as agents to mediate retroviral-mediated gene transfer into eukaryotic cells.
Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene ( 8 ) of interest. ~06t often, the structural genes (i.e., gag, pol, and env), are removed from the retroviral bA~-khnnP
using genetic ~n~i n~aring techni~ues known in the art.
This may include digestion with the appropriate restriction endonuclease or, in some instances, with 3al 31 exonuclease to generate fragments containing appropriate portions of the packaging signal.
These new genes have been incorporated into the proviral h~ckhnna in several general ways. The most straightforward constructions are ones in which the structural genes of the retrovirus are replaced by a single gene which then is transcribed under the control of the viral regulatory sequences within the long t~rminAl repeat (LTR). Retroviral vectors have also been constructed which can introduce more than one gene into target cells.
Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a 6pliced message or i8 under the regulation of it6 own, internal promoter.
Efforts have been directed at m;n;nl;7;ng the viral ^nt of the viral backbone, largely in an effort to reduce the chance f or recombination between the vector and the packaging-defective helper virus within packaging ,~ WO 95/15167 2 ~ 7 7 ~ ~ 8 PCTIIJS94113811 cells. A packaging-defective helper viru& is necessary to provide the structural genes of a retrovirus, which have been deleted from the vector itself.
In one: ~;r 1_, the retroviral vector may be one of a series of vector5 de5cribed in Bender, et al., J. Virol.
61:1639-1649 ~1987), based on the N2 vector (Armentano, et al., J. Virol., 61:1647-1650) containing a series of deletions and substitutions to reduce to an absolute minimum the homology between the vector and packaging systems . These changes have also reduced the 1; k.ol; h--od that viral protein6 would be expressed. In the f irst of these vectors, LNL-XHC, there was altered, by site-directed mutagenesis, the natural ATG start codon of gag to TAG, thereby eliminating unintended protein synthesis from that point. In Moloney murine leukemia virus (MoMuLV), 5 ' to the authentic gag start, an open reading frame exists which permits expression of another glycosylated protein (pPr80'~). Moloney murine sarcoma virus (MoMuSV) has alterations in this 5' region, including a fL hift and 1055 of glycosylation sites, which obviate potential expression of the amino terminus of pPr80i4. Therefore, the vector LNL6 was made, which incorporated both the altered ATG of LNL-XHC and the 5 ' portion of MoMuSV. The 5 ' structure of the LN vector series thus eliminates the possibility of expression of retroviral reading frames, with the subsequent production of viral antigens in genetically transduced target cells. In a final alteration to reduce overlap with packaging-def ective helper virus, Miller has eliminated extra env sequences immediately preceding the 3 ' LTR in the LN vector (Miller, et al., Biotechnigues, 7: 980-990, 1989 ) .
The paramount need that must be satisf ied by any gene transfer system for its application to gene therapy is safety. Safety is derived from the combination of vector genome structure together with the packaging system that is Wo 95/15167 21~ ~ ~ 9 8 PCT/US94~13811 utilized for production of the infectious vector. Miller, et ~ l . have developed the combination of the pPAM3 plasmid (the packaging-defective helper genome) for expression of retroviral structural proteins together with the LN vector series to make a vector pi~rk;~i ng system where the generation of r~ ' in~nt wild-type retroviru6 is reduced to a minimum through the elimination of nearly all sites of recombination between the vector genome and the packaging--defective helper genome (i.e. LN with pPaM3).
In one ' ' i r t, the retroviral vector may be a Moloney Nurine Leukemia Viru6 of the LN series of vectors, such as those hereinabove mentioned, and described further in Bender, et al. (1987) and Niller, et al. (1989). Such vectors have a portion of the packaging signal derived from a mouse sarcoma virus, and a mutated gag initiation codon.
The term "mutated" as used herein means that the gag initiation codon has been deleted or altered such that the gag protein or f ragments or truncations thereof, are not expressed .
In another: ~ nrli L, the retroviral vector may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average f requency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average DNA size of at least 10,000 base pairs. Preferred cloning sites are selected from the group consisting of NotI, SnaBI, SalI, and XhoI. In a preferred L~ the retroviral vector includes each of these cloning sites . Such vectors are further described in U. S .
Patent Application Serial No. 919,062, filed July 23, 1992, and incorporated herein by reference.
When a retroviral vector including such cloning sites is employed, there may also be~ provided a shuttle cloning vector which includes at least two cloning sites which are compatible with at least' two loning sites selected from WO 95115161 2 1 7 ~ 9 ~ 8 PCrlUS94/13811 the group consisting of NotI, SnaBI, SalI, and XhoI located on the retroviral vector. The Dhuttle cloning vector also includes at least one desired gene which is capable of being transferred from the shuttle cloning vector to the retroviral vector.
The shuttle cloning vector may be constructed f rom a basic "hArkh~)n~'~ vector or fragment to which are ligated one or more linkers which include cloning or re6triction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.
The shuttle cloning vector can be employe~ to amplify DNA sequences in prokaryotic systems. The shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria. Thus, for example, the shuttle cloning vector may be derived from plasmids such as pBR322; pUC 18; etc.
The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral I.TR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biot~rhniçues~ Vol. 7, No. 9, 980-990 (lg89), or any other promoter (e.g., cellular promoters such as eukaryotic r~el 1111Ar promoters including, but not limited to, the histone, pol III, and B-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TE~ promoters, and Bl9 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
The vector then is employed to transduce a packaging cell line to form a producer cell line. Examples of Wo 95/15167 PCT~'S94/13811 217 ~ ~9~ ~
parkA~ing cell6 which may be transfected include, but are not limited to, the PE501, PA317, -2, -AM, PAl2, T19-14X, VT-19-17-H2, CRE, CRIP, GP+E-86, GP+envAml2, PAT
2 . 4, and DAN cell lines . Representative examples of packaging cell lines al60 are described in Miller, Human Gene Ther~py, Vol . 1, pgs . 5-14 ( 1990~) . The vector containing the nucleic acid se~uence èncoding the therapeutic agent may transduce the parkA~i n~ cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0~ precipitation.
In one ` ~ L, a first packaging cell line, such as PE501, is transfected with the vector, and viral particles are generated. ~hese infectious viral particles then are used to transfect a second packaging cell line, such as PA317, which generates an increased amount of viral particles .
The producer cells are then administered n vivo to the f etus in an amount ef f ective to produce a therapeutic effect in the fetus. In general, the producer cells are administered to the f etus in an amount of f rom about l x lO' cells to about l x lO'~ cells, preferably from about 1 x 10' cells to about l x 109 cells, more preferably in an amount of from about 5 x lO' cells to about 1 x 10~ cells.
The producer cells may be administered to the f etus systemically, such as by intraperitoneal administration, intravenous administration, or by direct injection into an organ or muscle. ~he amount of producer cells to be administered is dependent upon various factors, including the disease to be treated and the extent and severity thereof .
The producer cells are administered in combination with a pharmaceutically acceptable carrier suitable f or administrati~n to a patient. The carrier may be a lis~uid Wo 95/15167 2 ~ 7 ~ g 9 ~ PCrlUS94/13811 carrier such as, for example, a saline solution or a buffer solution or other isomolar aqueou6 601ution.
Upon admini6tration of the producer cell6 to the fetus, the producer cells generate viral particle6. The viral particle6 then transduce fetal ceIls, such as, for example, hematopoietic stem cells, cells of the central - nervous system, and cells of other tissues and organs, whereby the transduced cells expres6 the therapeutic agent.
In another alternative, a viral supernatant containing viral particle6 may be administered to the fetu6, whereby such viral particles transduce fetal cells as hereinabove mentioned. The viral supernatant may be administered in an amount of from about l x 103 CEU to about l x lO" CFU, preferably from about l x 104 CF~ to about l x lOY CFU.
Therapeutic agents which may be encoded by the at least one nucleic acid sequence include, but are not limited to, those which treat hematopoietic system deficiencies, immune defin;~nci-~c, lysosomal storage disorders, Lesch-Nyhan disease, and leukocyte adhesion deficiency. Specific examples of therapeutic agents include, but are not limited to, Factor VIII, for treating h ,h;li~ A; Factor IX, for treating hemophilia B; FACC, for treating Fanconi anemia; a-globin, for treating a-thalassemia; B-globin, for treating B-thalassemia and sickle cell anemia; adenosine ~ m;nA~e (ADA), and PNP, for treating severe combined; 'eficiency; the T-cell receptor a-chain, for treating X-linked immunodeficiency;
glucocerebrosidase, for treating Gaucher's disease;
iduronate sulfatase, for treating Hunter's syndrome; a-L-iduronidase, f or treating Hurler ' s syndrome, a-galactosidase, for treating Fabry disease; the a-subunit of h~Yns;lm;n~ e A, for treating Tay-Sachs disease; HPRT, for treating Lesch-Nyhan disease; and CDl8 complex, for treating leukocyte adhesion def iciency . It is to be understood, however, that the scope of the present Wo 95/15167 2 1 7 ~ ~9 ~ PCrlUS94113811 ~
invention is not to be limited to the above-mentioned therapeutic agents.
In addition, the process of the present invention may be applied to fetal animals in order to suplly to the fetal ~nimal therapeutic agents such as growth hormones, and agents which confer resistance to disease. In addition, the process of the present invention may be employed to provide a fetal animal with a gene encoding a human protein or therapeutic agent. For example, a fetal animal may be given a gene for human hemoglobin, which then may be harvested from the animal after birth to provide a human blood substitute.
Also, the process of the present invention may be employed to provide animal models for gene expression and gene therapy.
The invention now will be described with respect to the following examples; however, the scope of the present invention is not intended to be limited thereby.
E:xam~le 1 Construction of DGlNaSvAd and ~ener~tion of producer cells and viral supernatant theref rom A. Construction of ~GlNaSvAd Plasmid pGlNaSvAd was derived from plasmid PGl (Figure 3). Plasmid pGl was constructed from pLNSX (Palmer, et al., ~3100d, Vol. 73, pgs. 438-445. The construction strategy for plasmid pGl is shown in Figure l . The l . 6kb EcoRI fragment, containing the 5 ' Noloney Murine Sarcoma Virus (MoMuSV) LTR, and the 3 . Okb EcoRI/ClaI fragment, containing the 3 ' LTR, the bacterial origin of replication and the ampicillin resistance gene, were isolated separately. A linker containing seven unique cloning sites was then used to close the EcoRI/ClaI f ragment on itself, thus generating the plasmid pGO The plasmid pGO was used ~ WO95/15167 2 i 77~8 PCT/US94/13811 to generate the vec, Jtor plasmid pG1 ( Figure 3 ) by the insertion of the 1.`6kB EcoRI fragment cofitaining the 5 ' LTR
into the unique EcoRI site of pGO. Thus, pGl (Figure 3) con6ists o$ a retroviral vector harkhr~p composed of a 5 ' portion derived from MoMuSV, a 6hort portion of gag in which the authentic ATG start codon has been mutated to TAG
~Bender, et al. 1987), a 54 base pair multiple cloning site (MCS) containing, from 5' to 3' the sites EcoRI, NotI, SnaBI, SalI, BamHI, XhoI, HindII, ApaI, and ClaI and a 3' portion of MoMuLV from base pairs 7764 to 7813 ( ' ed as described (Van Beveren, et al., Cold Spr;ng Harbor, Vol. 2, pg. 567, 1985) (Figure 2)~ The NCS was designed to generate a maximum number of unique insertion sites, based on a screen of non-cutting restriction enzymes of the pG1 plasmid, the neo' gene, the B-galactosidase gene, the IIYYL~ y-:in' gene, and the SV40 promoter.
The "backbone" vector pGlNa was constructed from pG1 and pN2 (Armentano, et al., J. Viroloav, Vol. 61, pgs.
1647-1650 ,~1987 ) ) . pGlNa was constructed by cutting pN2 (Figure 4) with EcoRI and AsuII, filling in the ends of EcoRI/AsuII fragment containing the neo0 gene, and ligating the fragment into SnaBI digested pG1 to form pGlNa (Figure 5) .
An SvAd fragment containing the human adenosine tle~mi n~e (ADA) cDNA sequence(Adrian, et al ., Mol . Cell .
~, Vol. 4, 1712-1717(1984); Wiggman, et al., ~, Vol.
80, pg. 7481(1983); Wiggman, et al., Nucl. Acids Res., Vol.
12, Pg. 2439 (1984) ) and promoted by an SV40 promoter (approximately 1.6 kb), wa6 cut from pB2SA with EcoRI and blunt ended with Klenow. (SvAd is available from other vectors that are available to those skilled in the art.
Preferably, it would be obtained from the vector SAX, described in Kantoff, et al., PNAS, Vol. 83, pgs. 6563-6567 (1986)) pGlNa was linearized 3' to the neoR gene by a double enzyme cut with SalI and ~ind III, and blunt ended .

WO 9S/15167 2 1 7 ~ ~ 9 ~ PCr/US94/13811 with Rlenow. The SvAd fragment then was ligated into pGlNa to ~orm pGlNaSv~d (Figure 7)J A schematic of the construction of pGlNaSvAd is shown in Figure 6.~
B. Gener~tion Qf PA317/GlNaSvAd Producer Cell 1ine A producer cell line was made from vector plasmid and packaging cells. The PA317/GlNaSvAd producer cell was made by the same techniques used to make previous clinically relevant retroviral vector producer cell Iines. The vector plasmid pGlNaSvAd DNA was transfected into a ecotropic packaging cell line, PE501. Supernatant from the PE501 transfected cells was then used to transinfect the amphotropic, hTR containing, pFIokil~;ng cell line ~PA317).
Clones of transinfected producer cells were then grown in G418 containing medium to select clones that contain the NeoR gene. The clones were then titered for retroviral vector production. Several clones were then selected for further testing and finally a clone was selected for clinical use.
s x 105 PE501 cells ~Miller, et al., Biotechni~ues, Vol . 7, pgs . 980-990 ~1989 ), incorporated herein by reference) were plated in 100 mm dishes with 10 ml high glucose Dulbecco's Modified Essential Medium ~DMEM) growth medium supplemented with 10% fetal bovine serum ~HGD10) per dish ~3-100 mm dishes are required per transfection). The cells were incubated at 37C, in a 5% CO2 atmosphere overnight .
The plasmid pGlNaSvAd then was transfected into PE501 cells by CaPO4 precipitation using 50 ~Lg of DNA by the following procedure.
50 ~g of DNA, 50 ~Ll 10 x CaCl2, and 450~1 of sterile H~O was mixed in a 15 ml polypropylene tube to yield a 0 . 25M Ca Cl2 solution containing 50 ~g DNA, 0 . 5 ml 2x PBS
( containing 50 mM N-N-bis- ( 2-1lydlu~sy~Lhyl ) - 2-~minoethane-sulfonic ~cid, 280 mM NaCl, 1.5 mM Na2HP04, and 50 mM Hepes, pH6 . 95 ), then was added to the tube and the contents of the ~ Wo95115167 2 1 7~9~ PCrrUss4J1381]
tube were mixed by pipetting. The DNA solution then was left at room temperature for about 20 minutes to 1 hour.
lml of ~NA solution then was added to each culture dish, and each dish was swirled to ensure even distribution of the DNA. The dishes then were incubated at 35C in a 3%
C02 atmo6phere overnight.
A culture dish(es) with optimum precipitate following the overnight incubation then was selected. The medium/DNA
precipitate was aspirated f rom the dish ( es ), and 5 mL PBS
was added to each dish. The dish(es ) was allowed to sit for 2 to 3 minutes to allow salts to dissolve.
The dish ( es ) then was washed again with PBS to remove the salt and the salt solution. 10 ml of HGD10 medium then was added to the dish ~ es ), and the dish ( es ) incubated at 37C in a 5% C0~ atmosphere for about 48 hrs.
A 48 hour transient supernatant then was collected from the transfected cells by removing the supernatant from the cells and placing it in a 15 ml polypropylene tube.
The dish ( es ) then was rinsed with 5 ml PBS . The PBS then was removed, and 1 ml trypsin-EDTA was added to each dish.
Three 15 ml polypropylene tubes then were labeled undiluted, 1:10, and 1:100, respectively. 9 ml of HGD10 plus 0 . 8 mg/ml of G418 were added to the 1:10 and the 1:100 tubes .
When the cells were no longer adherent to the dish, 9 ml of HGD10 and 0 . 8 mg/ml of G418 were added to the undiluted tube, and the cells transferred to the undiluted tube .
Serial dilutions of the cells then were made by adding 1 ml of undiluted cells to the 1:10 tube, and then by adding 1 ml of the 1:10 cells to the 1:100 tube. The cells then were mixed.
10 ml of HGD10 and 0 . 8 mg/ml G418 were added to each of six 100 mm dishes . To one dish was added 0 . 5 ml of u=d lute~ cells to make 1:2~ dilution o~ cells; to o=e 2~7~98 Wo 95/15167 PCTIUS94/13811 dish was added 0 . 25 ml of undiluted cells to make a 1: 40 dilution of cells; to one dish was ~dded 1.0 ml of the 1:10 dilution to make a 1:100 dilution of cells; to one dish was added 0.2 ml of the 1:10 dilution to make a 1:500 dilution of cells; to one dish was added 1. 0 ml of the 1:100 dilution to make a 1:1, 000 dilution of cells; and to another dish was added 0.5 ml of the 1:100 dilution to make a 1: 2, 000 dilution of cells .
The six plates of cells were ~ ; ned daily. The medium was changed if there was a great amount of cell death. Such medium changes were repeated until few dead cells were observed. At this point, live cells or colonies were allowed to grow to a size such that the colonies are large enough to clone out ( i . e ., the colonies are visible to the naked eye when looking up through the bottom of the plate). Viral supernatants from such colonies of PE501 cells were collected in amounts of from about 5 ml to about 10 ml, placed in ~:lyuLL~l~e6, and frozen in liquid nitrogen at about - 7 0 C .
PA317 cells (ATCC Accession No. CRL 9078 ) (l!~iller et al., Mol. Cell. Biol., 6:2895-2902 (1986)) and described in U.S. Patent No. 4,861,719, then were plated at a density of 5 x 104 cells per 100 mm plate on Dulbecco's Mor;ified Essential Medium (DMEM) including 4.5 g/l glucose, glutamine supplement, and 10~ fetal bovine serum (FBS).
The viral supernatant then was thawed, and 8 ~ug/ml of polybrene was added to viral 6upernatant from PE 501 cells, and the supernatant and polybrene were mixed and loaded into a syringe with a 0 . 22 ~m f ilter unit . The DMEM was suctioned off the plate of cells, and 7 to 8 ml of viral supernatant was added f or overnight inf ection .
The viral 6upernatant then was removed and replaced with fresh 1096 FBS. ûne day later, the medium was changed to 10~; FBS and G418 ( 800 ~g/ml ) . The plate then was monitored, and the medium was changed to fresh 1096 FBS and ~ WO 95115167 2 1 7 7 ~ 9 ~ PCT/US94~13~11 G418 to eliminate dying or dead cells whenever necessary.
The plate also was monitored for at least 10 to 14 days ~or the appearance of G418 resistant colonies by scanning the bottom of the di6h without a microscope. When colonies are large enough to see, they then were selected as clones.
The medium then was aspirated f rom the dish and replaced with 5 ml PBS. The cells then were rinsed and most of the PBS was aspirated. About 0 . 5 to 1. 0 ml of the PBS was left on the plate to keep it moist. Cloning rings then are placed on ell selected colonies. Two drops of trypsin-EDTA then 2 were placed on each cloning ring. The dish then was placed in an incubator, and tapped periodically until the cells are released from the dish. 5 ml of HGD10 plus 0 . 8 mg/ml was added to as many wells as needed in six well dishes.
When the cells from each colony were released from the dish, 2 drops of HGD10 are added to each cloning ring. A
pipette then was set to 200 ~1, and inserted into a cloning ring in order to remove all the cells. The cells then were transferred to one of the wells in the 6-well dishes. Such procedure was repeated until all desired clones were picked. The 6-well dishes were incubated at 37C in a 59 CO2 atmosphere.
The clones then were observed for confluent growth.
When a clone was confluent or almost conf luent, the clone was trypsinized and ~ n~iPd in a 100 ml dish.
When the ~nrl(-d clone was about 9096 confluent, the old medium was removed and replaced with 10 ml of fresh HGD10 medium. The dish was returned to the incubator for 2 0 to 2 4 hours .
After the incubation, the supernatant was removed from the dish, and placed in a 15 ml polypropylene tube. The tube was centrifuged at 1,200 to 1,500 rpm for 5 minutes to pellet out any cells which may have been in the supernatant .

O95115167 2 ~ 9 8 PCT/US94/13811 The aupernatant then was aliquoted into six cryovial6 ~1 mlJvial ) . The aliq~lots were stored in liquid nitrogen .
5 ml of PBS were added to the dish and the cells were rin6ed .
When the cells were released from the dish, 9 ml of HGD10 was added to the cells, and the cells were trans~erred to a 15 ml polypropylene tubè. The cells were pelleted by centrifuging at 1,200-1,500 rpm for 5 minutes.
The medium was aspirated o~f the cell pellet. The pellet then was resuspended in lml ~GD 10 and 1 ml of 2xDMSO freezing medium, and 1 ml of cells was aliquoted into each of two cryovials. The cryovials were placed on dry ice, and, when frozen, were transferred to liquid nitrogen .
Through the above procedures, a clone with the highest titer, designated as producer cell line PA317/GlNaSvAd.24, was used to produce a master cell bank of producer cells.
C . Production of Vir~ 1 S-~ernatant Which have been Cryopreserved PA317/GlNaSvAd.24 cells are thawed at 37C and recultured as rapidly as possible to avoid damage to the cells by the cryopreservative dimethylsulfoxide (DMSO).
The contents of one working cell bank cryovial are placed in a T75 flask containing 25 ml of medium containing high glucose (4.5g/1) DMEM with 10% FBS and 2mM
glutamine. The contents of this flask then are split into 4 to 10 75 cm2 f lasks . The contents of these flasks then can be split into additional 75 cm2 flasks. When the cells are about 90% confluent, the medium in all the flasks is changed .
Supernatant then is collected from the flasks, and pooled in a large 6pinner flask. A sample is taken for NYco~lasma screening. The rc--;nin~ supernatant is filtered through a 0.22 micron filter, pooled (samples ~ WO 95/1~167 2 t 7 7 ~ 9 g PCT/lJS94/13811 taken for further lot release te6ting), and aliquoted into the final containers. Nore medium i5 added to each flask.
One day later, 6upernatant again i6 collected from the flasks and pooled and filtered as hereinabove described. The procedure again is repeated the next day.
After the supernatant is aliquoted and labeled, it is stored in a freezer at -70C.
Example 2 Construction of pGlTkSvNa Anr~
çeneration of producer cell line therefrom A. Constructio~ of DGlTkSvNa The following describes the construction of pGlTkSvNa, a schematic of which is shown in Figure lO.
This vector contains the Thymidine Kinase ~hTK) gene from Herpes Simplex Virus I regulated by the retroviral promoter and the bacterial gene, neomycin phosphotransferase (NeoR) driven by an SV40 promoter. The hTK gene confers sensitivity to the DNA analogs acyclovir and ~AnrirlQvir while the NeoR gene product confer resistance to the neomycin analogue, G4l8.
To make pGITkSvNa, a three step cloning strategy was used. First, the herpes simplex thymidine kinase gene ( Tk ) was cloned into the Gl plasmid backbone to produce pGlTk. Second, the NeoR gene (Na) was cloned into the plasmid pSvBg to make pSvNa. Flnally, SvNa was excised from pSvNa and ligated into pGlTk to produce pGlTkSvNa.
pGl was constructed as desc,7ibed in Example l.
To construct pBg ( Figure 8 ) the 3 . O kb BamHI/EcoRI lacZ fragment that encodes ,~-galactosidase was isolated from pMCl871 (phArr~ ). This fragment lacks the extreme 5 ' and 3 ' ends of the ,B-galactosidase open reading frame. Linkers that would restore the complete lacZ open WO 95115167 217 ~ ~ ~ 8 pcrNs94ll38ll reading frame and add restriction sites to each end of the lacZ gene were synthesized and ligated to the BamHI/EcoRI
lacZ fragment. The structure of the 5 ' linker was as follows: 5' - 1/2 NdeI - SphI - NotI - SnaBI - SalI - SacII -AccI - NruI - BgIII - III 27 bp ribosomal binding signal -Kozak consensus sequence/NcoI - first 21 bp of the lacZ
open reading frame - 1/2 BamHI - 3 ' . The structure of the 3 ' linker wa6 as follows: 5 ' - 1/2 mutated EcoRI - last 55 bp of the lacZ open reading frame - XhoI
- HindIII - Smal - 1/2 EcoRI - ~3 ' . The restriction sites in the linkers were chosen because they are not present in the neomycin resistance gene, the ,~-galacto6idase gene, the l~yyL~ y~:in resistance gene, or the SV40 promoter. The 27 bp ribosomal binding signal was included in the 5 ' linker because it is believed to enhance mRNA stability (Hagenbuchle, et al., Cell 13:551-563, 1978 and Lawrence ~md Jackson, J. Mol. Biol. 162:317-334, 1982). The Kozak consensus sequence (5 '-GCCGCCACCATGG-3 ' ) has been 6hown to signal initiation of mRNA translation (Kozak, Nucl.Acid6 Res. 12:857-872, 1984). The Kozak consensus sequence includes the NcoI site that marks the ATG translation initiation codon.
pBR322 (Bolivar et al. Gene 2:95, 1977) was digested with NdeI and EcoRI and the 2 .1 kb f ragment that contains the ampicillin resistance gene and the bacterial origin of replication was isolated. The ligated 5 ' linker - lacZ - 3 ' linker DNA described above was ligated to the pBR322 NdeI/EcoRI vector to generate pBg. pBg has utility as a shuttle plasmid because the lacZ gene can be excised and another gene inserted into any of the restriction sites that are present at the 5 ' and 3 ' ends of the lacZ gene. Because these restriction site6 are reiterated in the pGl plasmid, the lacZ gene or genes that WO 95/15167 2 ~ 7 7 ~ 9 8 PCTIUS94113811 replace it in the shuttle plasmid construct can easily be moved into pG 1 .
A 1. 74 kB BglII/PvuII fragment containing the Herpes Simplex Virus Type I thymidise kinase gene (GenBank accession no. V00467, incorporated herein by reference) was excised from the pXl pla6mid (Huberman, et al., ExPtl. Cell 1~ Yol. 153, pg6 347-362 (1984) incorporated herein by reference), blunted with the large (Klenow) fragment of DNA
polymera6e I, and in6erted into the unique SnaBI 6ite in the pGl mul,~iple cloning 6ite, to form pla6mid pGlTR.
(Figure 9 ) .
A 339 bp PvuII/HindIII SV40 early promoter f ragment obtained f rom the pla6mid pSV2Neo ( Southern et al, Jol~rnal of ~5olecular and ADDlied Genetic6 1:327-341(1982) ) wa6 then in6erted into psg in the unique NruI 6ite to generate the pla6mid pSvBg ( Figure 5 ) . The pSvBg pla6mid was dige6ted with BglII/XhoI to remove the ~ gene, and the end6 were made blunt u6ing the Rlenow fragment. An 852 bp EcoRI/AsuII fragment containing the coding 6equence of the neomycin resi6tance gene wa6 removed from pN2 (Armentano, et al., J. Virol., Vol. 61, pg6. 1647-1650 (1987) ), blunted with Rlenow fragment and ligated into the 2.5 kb blunted BglII/XhoI fragment generated hereinabove, re6ulting in pSvNa. The SV40 promoter/neomycin re6i6tance gene ca66ette wa6 then removed from pSvNa a6 a ll91bp SalI/HindIII fragment. The pGlTk plaamid was then digested with SalI/HindIII and ligated with the SV40/neor fragment to generate pGlTkSvNa. (Figure 10) ~
B . Generation of Producer Cell l,ine PAT 2 . 4/GlTkSV
Na. 90 ;Inr~ Generation of Viral SuDernatant Theref ~om .
Producer cell line PAT 2 . 4/GlTkSvNa. 90 was prepared according to the method disclosed in Example 1 for wc 95~l5~67 2 1 ~ ~ 9 ~ ~ PCr/USs4/13811 the preparation of producer cell line PA317/GlNaSvAd.24 except that rAI-kA~i n~ cell line PAT 2 . 4 was used instead of p~rk~ing cell line PP.317. PAT 2.4 was made according to the method for the preparation of PA317 cells disclosed in Miller et al., Mol. Cell. Biol., 6:2895-2902 (1986) and in U.S. Patent No. 4,861,719 issued August 29, 1989 to ~iller, the disclosure o~ which is incorporated herein by reference, except that the NIH 3T3 TK-minus cells were co-transfected with the llyyL~ ycin resistance gene instead of the Herpes Simplex Virus thymidine kinase gene to provide a selectable marker and the p~ck~gi n~ cell line was selected from oligo-clonal populations.
The method is summarized as follows. A
population of the NIH 3T3 TK-minus cells was co-transfected with two plasmid DNAs at a ratio of 20 :1 using standard C~PO4 transection methodology . The f irst plasmid was pPAM3 (ATCC accession number 40234 ), which contains the promoter, gag, pol, and env sequences of amphotropic murine leukemia virus. Seventy micrograms of DNA were used. The second plasmid was pY3, which contained the ~IyyL~ ycin resistance gene. (The ~IyyLI y~:in resistance gene also is found in other plasmids which are available to those skilled in the art. ) The population of cells was selected in IIYYL~ Y~:in, and llyyL~ y~:in resistant cells were frozen as primary and secondary seed lots. The population was analyzed for viral envelope expression, and the rArk~in~ function was tested in TK vector producer cell clones to look for high titer vectors. Oligo-clonal populations were created by seeding one 24-well plate with 5-10 cells per well and another plate with 10-20 cells per well. A total of 46 populations were created. Each oligoclone was ~ n-i~d and frozen.
Each population was tested individually for packaging ef f iciency by generating a producer population with supernatant from the packaging cell PE501, which had been transfected with the plasmid pGlTkSvNa, containing the WO 95/15167 2 1 7 7 ~ g ~ PCrlUS94~13811 neomycin resistance gene according to the procedure of Example 1. A subpopulation capable of producing high titer producer cell lines was identified according to the procedure of Example 1 and designated PAT 2 . 4/GlTkSvNa. 90 .
Viral supernatant containing GlTkSvNa was prepared from this producer cell in accordance with Example 1.
r le 3 A. Gene Transfer into Sheep Fetuses Ten ewes with ~onf; ~ dates of pregnancy were prepared for surgery by withholding food for 48 hours and water for 18 to 24 hours. Each ewe was sedated with intramuscular ketamine ~lOmg/kg), and given a 0.5 to 1096 halothane-oxygen inhalation mixture via an endotracheal tube. Each animal received intravenous fluids and antibiotics during surgery. The uterus was exposed by a lower midline incision, and each fetus was accessed through a small hysterotomy and transverse incision of the y~ Lrium and chorion . The amnion was lef t intact . The fetus then was visualized, and gently manipulated into an amniotic bubble. The fetus then was immobilized for injection within the bubble under gentle applied pressure.
At this time, the fetus is within view fully, which insures that the injected cells or viral supernatant remain within the peritoneal cavity. The sheep were injected with one of the following: (i) 2 ml of viral supernatant from GlNaSvAd.24, having a titer of 1 x 10' CFU/ml; (ii) 1 ml of 5 x 107 PA317/ GlNaSvAd.24 producer cells; (iii) 1 ml of 5 X 107 PA317/GlNaSvAd. 24 producer cells that were irradiated with 3,000 rads in a cesium irradiator; (iv) 2 ml containing 1 x 105 PAT2.~/GlTkSvNa.90 producer cells; (v) 1 ml of 5 x 107 PAT2.4/GlTkSvNa.90 producer cells; (vi) 2 ml containing of 1 x 105 PAT2.4/GlTkSvNa.90 producer cells that were ir adiated with 3,0~ rads in a cesium WO 95/15167 ~ 9 9 ~ PCrn,'S94/13811 irradiator; (vii) 1 ml of 5 x 10' PA317 pP~~kP~ing cells;
(viii) 1 ml of 5 x 107 PAT2.4 packP~ins cells; or (ix) 2 ml containing 1 x 10~ PAT2.4 packaging cells. The fetuses of each ewe were injected with viral supernatant or cells a6 ahown in Table 1 below.
. .
Aqe of ~m~
~r~rimental Number of fetu6 of mls Sheep Group f etu6e6 ( day ) i n jected 1.GlNaSvAd . 24 2 672 ml supernatant 2 .GlNaSvAd . 24 1 672 ml 6upernatant 3.PAT2.4/GlTkSvNa.90 2 672 ml producer cell6 4 .PAT2 . 4/GlTkSvNa . 90 2 672 ml producer cells 5 .P~T2 . 4/GlTkSvNa . 90 1 672 ml irradiated producer cells 6.PAT2.4/GlTkSvNa.90 2 672 ml irradiated producer cells 7.PAT2.4/GlTkSvNa.90 1 672 ml Wo 9S/15167 21 7 ~ 9 ~ ~ PCTIUS94/~3811 irradiated producer cells 8. PA317/GlNaSvAd.24 1 571 ml producer cells 9. PA317/GlNaSvAd.24 2 571 ml producer cells 10. PA317/GlNaSvAd.24 3 571 ml irradiated producer cells After the injection, each fetus was returned to the primary amniotic space, and the myometrium was closed in a double layer. Following the closing of all incisions, each ewe was observed for 48 hours and, unless scheduled for further experimentation, was returned to a large animal facility for the 1. -;nrl~r of the gestation period. Each ewe that survived was examined one week before the expected date of delivery. (Normal gestation is 145 d~ys. ) Of the 17 fetu6es injected, 3 were born alive that were injected with GlNaSvAd.24 viral supernatant; 2 were born alive that were injected with non-irradiated PAT 2.4 GlTkSvNa.90 producer cells; and 4 were born alive that were injected with irradiated producer cells(2 with PA317/GlNaSvAd.24 irradiated producer cells and 2 with PAT 2 :4/GlTkSvNa. gO
irradiated producer cells ) .
B. Assav Proc~hlres ~or Det-rrinin~ Presence of neo~
~nl1 ~erpeg Si lex Thymil1ine E~in~e Genes At various points after birth, CFU-E, BFU-E, CFU-GM, or CFU-Mix bone marrow cells (all of which are hematopoietic stem cells ) were taken from various sheep and Wo 9~ 167 217 f ~ 9 ~ PCr/US9~113811 analy~ed for the expression of the neo~ gene by culturing the cells in the pre6ence of G418, and/or for expression of the Herpes Simplex thymidine kinase (TR) gene by culturing the cells in the presence of ganciclovir.
CFU-E cells and BF~-E cells were tested in a plasma clot culture assay as iollows:
Whole bone marrow from the sheep was transferred to a 15 ml centrifuge tube, and the tube was spun at l, 600 rpm for lO minutes. The buffy coat wa6 removed and the marrow cells brought up to 15 ml with Iscove ' s solution .
The cells then were layered onto 5 ml of Ficoll buffer and spun for 30 minutes at 2,000 rpm. The mononuclear layer is removed and the ~ i n i nq cells are washed twice with Iscove ' s solution . The cells were resuspended in 5 ml of Iscove ~ s solution and counted . A medium containing the following, ~ ^ntS then was formed for each plasma clot culture:
100111 L-asparagine 300 ILl Fetal calf-serum lO0 ~l alpha-thiocyanate B
30 ~l erthyropoietin lO0 ~l 4 x Thrombin 630 f~l of this mix was pipetted into each tube.
The volume of cells was calculated that is needed to get 2 x lOs cells . G418 in amounts of 0 mg/ml, 0 . 5 mg/ml, l . 0 mg/ml, l . 5 mg/ml, 2 . 0 mg/ml, 2 . 5 mg/ml, or 3 . 0 mg/ml, and/or 0 ~M, 3~1M or 6~M of ganciclovir was added to each tube. Ganciclovir interacts with Herpes Simplex thymidine kinase in order to kill cells which express the Herpes Simplex thymidine kinase gene . The amount of Iscove ' s solution for each tube was calculated to bring the final volume of the tubes, including the cells, to l ml. The Iscove's solution was added to the tube, followed by the G418 and/or the ganciclovir, followed by the cells.

~ WO95115167 2177~ PCT/llS94/13811 100 ~-1 of citrated plasma, which aids in clotting, is added to the f irst tube . The contents of the tube are mixed by pipetting the contents up and down with a 1 ml pipette, and 200 ~Ll of the contents is dispensed to each of eight plasma clot wells. Four wells are in one 6 well plate, and four wells are in another 6 well plate.
The above procedure is repeated for all samples.
Water is added to the spaces in between the wells on the six well plate, and the plates are placed in the incubator until the cells are ready for harvest.
In order to harvest the clots, the plates are removed from the incubator, and allowed to sit under a hood for lO minutes. During this time, P03 buffer i8 placed into a petri dish.
Coated slides then are set up on a cafeteria tray and labeled. Four clots are lifted out for each group onto the same slide, and are arranged in an offset pattern so that the clots do not come in contact with each other.
A filter paper then is dipped into the P03 buffer, and a piece is laid over each slide. The filter papers and the 61ide6 are allowed to sit for l to 2 minutes .
A large piece of Baxter filter paper then is obtained and laid over all the slides. The paper is pres6ed firmly to blot out all of the excess buffer. The large filter paper is discarded, and 3~ glutaraldehyde is squirted onto each slide's P03 buffer coated filter paper.
The slide and filter paper are allowed to sit for l minute.
The slide's filter papers are removed and di6carded. The slides are set in a drying rack, and are allowed to dry for several hours before staining. Each slide then was treated with methanol for one minute, and then treated with l96 benzid'lle in methanol for 5 minutes. Each slide then was treated with peroxidase f or 3 minutes, and then washed with Wo 95/15167 2 1 7 7 ~ ~ 8 PCr~S94113811 with hematoxylin for 8 minutes, and then placed under cool running tap water $or lO minutes.
CFU-GM and CFU-~ix colonies were evaluated for neomycin resistance or expression of the Herpes Simplex thymidine kinase gene through a methylcellulose culture assay as follows-Bone marrow aspirates were drawn from sheep andmononuclear cells were isolated by layering lO ml of bone marrow (diluted 1:3 in IMDM) onto a cushion of 5 ml of Ficoll-Hypaque buffer. The tubes were subjected to centrifugation at 1,500 rpm for 30 minutes. The mononuclear fraction was removed with a sterile transfer pipette and washed twice with IMDM. The cells were pelleted and cultured at 2 x lOs cells/ml. Each plate consisted of 3 wells, each containing one-third of the following mixture:
1.6 ml of methycellulose (Gibco) 60 ~l of erythropoietin (50 unitsJml final concentration) 100 ~Ll of sheep phytohemagglutinin-stimulated leukocyte conditioned medium (PHA-LC~q) 4 x lO' cells G418 and/or ganciclovir IMDM to make a final volume of 2 ml G4 l 8 was added to the plates at concentrations of 0 mg/ml, 0 . 5 mg/ml, l . 0 mg/ml, l . 5 mg/ml, 2 . 0 mg/ml, 2 . 5 mg/ml, and 3.0 mg/ml, and/or ganciclovir was added at 0 f~rq, 3 IILM, or 6 ~M. These plates then were cultured for 7 days (CFU-GM), and 12 days (CFU-Mix), and colonies were counted under a dissecting scope.
r le 4 The effect of G41B on colony formation was evaluated, according to the assay procedures of Example 3, with respect to CFU-Mix, BFU-~, CFIJ-GM, and CFU-E cells 095/15~67 ~ 9~8 ~Cr/l~S94/13811 taken from six newborn control sheep that were not given producer cells or viral 6upernatant. The total number of colonie~ for each type of cell taken from the six sheep was calculated. The results are given in Figure 11. These sheep also were evaluated at 7 months and 17 months after birth. The results at 7 months and 17 months after birth were the same as when the sheep were newborn.
ExamDle 5 The effect of G418 on CFU-Mix, BFU-E, CFU-GM, and CFU-E cells of three of the sheep which were born alive, and now at ? months of age (10 months after intraperitoneal administration of viral supernatant or producer cells ), was evaluated and compared with the effect of G418 on the CFU-Mix, BFU-E, CFU-GM, and CFU-E bone marrow cells of six control sheep which were not given any producer cells or viral supernatant. In this experiment, one of the three treated sheep received GlNaSvAd.24 viral supernatant, one sheep received 5 x 10' irradiated PA317/GlNaSvAd.24 producer cells, and 1 sheep received 5 x 10' PAT2.4/GlTkSvNa.90 producer cells. For each sheep, the percentage of G418 resistant colonies of each cell type was det~rmin~l according to the assay procedures of Example 3, and the average percentage of G418 resistant colonies was calculated for each sheep. An average percentage of G418 resistance then was calculated for each group (control or treated~ of sheep for G418 in concentrations o~ O mg/ml, O . 5 mg/ml, 1. 0 mg/ml, 1. 5 mg/ml, 2 . O mg/ml, 2 . 5 mg/ml, and 3 . O mg/ml . The percentage of G418 resistant colonies in the control sheep and treated sheep at varying doses of G418 is given in Figure 12.~

Wo 95/15167 PCT~7S94/13811 2 ~ 9 8 le 6 Four sheep at age 6 months were evaluated for the presence of G418-resistant CFU-E, BFU-E, and CFU-GM cells in the pre3ence of 2 mg/ml G418 according to the assay procedures of Example 3. Sheep 1 received 5 x 107 P~T2 . 4/GlTkSvNa. 90 producer cells . Sheep 2 received 1 x 108 PAT2.4/GlTkSvNa.90 producer cells. Shèep 3 received 1 ml of 1 x lO' CE~U~'ml of GlNaSvAd.24 viral supernatant, and Sheep 4 received 5 x 107 irradiated PA3i7/GlNaSvAd. 24 producer cells. The percent resistance of the cells from each sheep to 2 mg/ml G418 is given in Table II below:
T 1~ RT .1;: I I
% resis7-~nce to G418 CFU--E _ BFIJ--E CFU-Grl Example 7 All the sheep that were born alive, which received producer cells, which produce viral particles expressing the neo~ gene, and all sheep that were born alive which received viral supernatant, were monitored for a period of 20 months for G418=resistant colonies of bone marrow cells according to the ass~ys hereinabove mentioned in Examples 3 and 5. The average percent of G418-resistant cells for all cell types, and then for all sheep which received viral supernatant or producer cells, was Wo 95115167 2 1 7 7 ~ ~ ~ PCTIIIS94/13811 calculated. The re6ults of such monitoring are shown in Figure 13. ~
Example 8 The effect of G418 and ganciclovir (GCV) on BFU-E
and CFU-GN cells was measured for Sheep 1 in Table II above according to the assay procedures mentioned above in Example 3. The test cells were contacted with G418 in a concentration of 3 mg/ml, and/or ganciclovir in an amount of 6 ~M. The results are shown in Figure 14 . ' As can be seen from Figure 14, the difference between the amount of colonies when neither G418 nor ganciclovir is administered, and when ganciclovir is administered is essentially equal to the number of G418-resistant colonies. Thus, it is implied that ganciclovir kills all the cells that are neomycin-resistant. When G418 and ganciclovir are present in the medium, esser,tially no cell colonies are formed, demonstrating that the cells resistant to G418 are sensitive to ganciclovir.
Exam~le 9 The percentage of neoR expression in all sheep which were born alive, and which were given producer cells carrying the neoR gene or viral supernatant, was monitored with respect to CFU-Mix, CFU-GM, BFU-E, and CFU-E cells for a period of 20 months. Average percentages of G418-resistance for each type of cells for all sheep were calculated, and the results are given in Figure 15.
Example 1 0 One sheep that received 5 x 107 irradiated PA317/GlNaSvAd.24 producer cells was sacrificed 13 months after injection of the producer cells (or 10 months after birth). PCR as6ays then were cnnFIl~tefl on DNA isolated from the lung, liver, kidney, and testes for presence of the neoR gene. The PCR assays were conducted as follows:
300 nanograms of total genomic DNA was subjected to analysis by the techni~ue of the polymerase chain Wo 95/15167 PCr/US94/13811 2177~98 reaction ~PCR) as described in Saiki, et al., Science, Vol. 230, pgs. 1350-1354 (1985) with the following ch~nges to the reaction constituents: the PCR primers used were 0 . 5 ~M of primer 1 and 0 . 5 ~LM of primer 2 . dATP, dCTP, and dGTP were used at 150 /lM. In order to eliminate the chance of product carry-over leading to false-positives, dUTP (600 ~LM) was substituted for dTTP, and reactions were treated with uracil-DNA-glycosylase (UDG) prior to amplification, according to the method of Wang, et al. American Journal of Hematoloav, Vol. 40, pgs. 146-148 t1992). MgCl~ was used at 5 mM, and 2.5 units of Amplitaq DNA polymerase (All PCR
reagents were purchased from Perkin Elmer. ) were added to each 50 ~l reaction. Primer 1 has the following sequence:

Primer 2 has the f ollowing sequence:
5 '-ATC CTG ~TC GAC AAG ACC GGC TTC--3 ' These primers amplify a 440 base pair fr~lgment of the neomycin resistance gene. The samples were overlaid with 100 ~l of mineral oil, heated to 95C for 10 minutes to inactivate the UDG, and then were subjected to 40 cycles of PCR. The reactions were run in an automated PCR
temperature cycling block that allowed denaturation of the DNA at 95C for 1 minute, ~nne~l ing of the primers at 65C
for 1.5 minutes, and extension of the primers at 72C for 1.5 minutes, and extension of the primers at 72C for 1.5 minutes. After the 40th cycle, the reactions were held at 72C to allow complete extension of the amplification products and to prevent damage due to residual IJDG
activity . Chlorof orm extractions were perf ormed on the reactions, and 15 ~l of each resultant aqueous pha6e was loaded onto a 29a agarose gel and electrophoresed in Tris-acetate-EDTA .
PCR analysis of DNA f rom the testes and kidney showed that both tissues contained the neoR gene; however, presence of the provirus in the kidney and testes genomes WO 95/15167 2 ~ 7 ~ 9 ~ 8 PCT~US94~138ll did not appear to have any deleteriou6 effects on the animal, as such organs showed no evidence of any pathologic condition upon examination. Such results indicate that direct in jection of an ~n~; ne~red retrovirus into a fetus is a feasible means of delivering a foreign gene to a developing fetus and achieving long term expression without endangering the recipient.
Example l l Another sheep that received 5 x lO~ irradiated PA317JGlNaSvAd.24 producer cells was sacrificed 18 months after injection of the producer cells (or 15 months after birth). A PCR assay then was conducted on DNA isolated from the brain for presence of the neoR gene. The PCR
assay was conducted as described in Example lO.
PCR analysis of DNA from the brain showed that brain cells contained the neoS gene. Thus, it has been shown that direct injection of an engineered retrovirus into a f etus can deliver a gene to the brain of a developing fetus.
All articles cited herein are hereby incorporated by ref erence .
It is to be understood, however, that the scope of the present invention is not to be limited to the specific ~ nts described above. The invention may be practiced other than as particularly described and still be within the ~ope of the ~ccomp :nyinl~ c1aim~ .

Claims (12)

WHAT IS CLAIMED IS:

1. A process for effecting gene therapy in vivo in a fetus, comprising:
transducing fetal cells in vivo with at least one nucleic acid sequence encoding a therapeutic agent.

2. The process of Claim 1 wherein said fetal cells are transduced in vivo with a viral vector including said at least one nucleic acid sequence encoding a therapeutic agent.

3. The process of Claim 2 wherein said viral vector is a retroviral vector.

4. The process of Claim 3 wherein said in vivo transduction of fetal cells is effected by administering to said fetus producer cells which generate said retroviral vector.

5. A process for effecting gene therapy in vivo in a fetus, comprising:
transducing fetal cells in vivo with a retroviral vector including at least one nucleic acid sequence encoding a therapeutic agent.

6. The process of Claim 5 wherein said in vivo transduction of fetal cells is effected by administering to said fetus producer cells which generate said retroviral vector.

7. The process of Claim 3 wherein said in vivo transduction of fetal cells is effected by administering to said fetus a viral supernatant which contains said retroviral vector.

8. The process of Claim 5 wherein said in vivo transduction of fetal cells is effected by administering to said fetus a viral supernatant which contains said retroviral vector.

9. The process of Claim 4 whereln said producer cells are administered intraperitoneally.

10. The process of Claim 6 wherein said producer cells are administered intraperitoneally.

11. The process of Claim 7 wherein said viral supernatant is administered intraperitoneally.

12. The process of Claim 8 wherein said viral supernatant is administered intraperitoneally.