a stable murine based rd114 retroviral packaging line efficiently transduces human hematopoietic cells

ARTICLE doi:10.1016/S1525-0016(03)00263-6 A Stable Murine-Based RD114 Retroviral Packaging Line Efficiently Transduces Human Hematopoietic Cells Maureen Ward,1 Rose Sattler,1 I Robert Grossman,2 Anthony J Bell, Jr.,1 Donna Skerrett,3 Laxmi Baxi,4 and Arthur Bank1,2,* Department of Genetics and Development, 2Department of Medicine, 3Department of Pathology, and 4Department of Obstetrics and Gynecology, Columbia University, New York, New York 10032, USA *To whom correspondence and reprint requests should be addressed at the Departments of Medicine and Genetics and Development, Columbia University, 701 W 168th Street, Room 1604, New York, NY 10032 Fax: (212) 923-2090 E-mail: ab13@columbia.edu Several barriers exist to high-efficiency transfer of therapeutic genes into human hematopoietic stem cells (HSCs) using complex oncoretroviral vectors Human clinical trials to date have used Moloney leukemia virus-based amphotropic and gibbon ape leukemia virus-based envelopes in stable retroviral packaging lines However, retroviruses pseudotyped with these envelopes have low titers due to the inability to concentrate viral supernatants efficiently by centrifugation without damaging the virus and low transduction efficiencies because of low-level expression of viral target receptors on human HSC The RD114 envelope from the feline endogenous virus has been shown to transduce human CD34؉ cells using transient packaging systems and to be concentrated to high titers by centrifugation Stable packaging systems have potential advantages over transient systems because greater and more reproducible viral productions can be attained We have, therefore, constructed and tested a stable RD114-expressing packaging line capable of high-level transduction of human CD34؉ cells Viral particles from this cell line were concentrated up to 100-fold (up to107 viral particles/ml) by ultracentrifugation Human hematopoietic progenitors from cord blood and sickle cell CD34؉ cells were efficiently transduced with a NeoR-containing vector after a single exposure to concentrated RD114-pseudotyped virus produced from this cell line Up to 78% of progenitors from transduced cord blood CD34؉ cells and 51% of progenitors from sickle cell CD34؉ cells expressed the NeoR gene We also show transfer of a human ␤-globin gene into progenitor cells from CD34؉ cells from sickle cell patients with this new RD114 stable packaging system The results indicate that this packaging line may eventually be useful in human clinical trials of globin gene therapy Key Words: RD114 envelope, retroviral packaging line, hematopoietic cell gene therapy INTRODUCTION Stable oncoretroviral packaging systems have been successfully used to introduce genes safely into human hematopoietic progenitor cells [1,2] These systems employ mouse fibroblast cells to express the gag and pol retroviral elements of the Moloney murine leukemia virus (MLV) as well as a viral envelope protein such as the amphotropic MLV-A [3] or the gibbon ape leukemia [4] virus (GALV) envelope These packaging systems have been used in human clinical trials to transfer human genes into hematopoietic stem cells (HSCs) [5– 8] An alternative envelope, which has been pseudotyped to both MLV [9] and the HIV-1 lentivirus [10], is the G-protein of the vesicular stomatitis virus (VSV-G) This envelope enables the viral particles to be concentrated by ultracentrifugation [11] 804 However, the VSV-G protein is toxic and therefore difficult to use in a stable packaging system Stable packaging systems are desirable because they permit the reproducible generation of large quantities of virus that can easily be tested for wild-type virus production compared to transient systems The available stable MLV-A and GALV packaging systems are limited by the low expression of receptors for these envelopes on human HSC [12–14] Another drawback has been the inability to make high-titer viral supernatants (greater than 106 viral particles/ml) from these stable cell lines, particularly when using complex retroviral vectors containing regulatory elements, such as the human ␤-globin gene [15,16] One strategy to overcome some of these drawbacks is to use an alternative viral envelope that more efficiently MOLECULAR THERAPY Vol 8, No 5, November 2003 Copyright © The American Society of Gene Therapy 1525-0016/03 $30.00 doi:10.1016/S1525-0016(03)00263-6 ARTICLE FIG Retroviral constructs used to make the RD114 stable packaging line (A) Vector expressing the Moloney leukemia virus gag and pol genes in the GP101 host 3T3 cell line (B) RDF: Vector expressing the RD114 feline endogenous viral envelope (C) SINPGKNeo: Self-inactivating retroviral vector containing a NeoR gene controlled by a PGK promoter (D) p141: Self-inactivating retroviral vector containing the human ␤-globin gene with a 372-bp deletion in IVSII and HS234 regulatory elements in the reverse orientation and the NeoR gene controlled by a PGK promoter targets HSC RD114 is an endogenous feline type C virus that readily infects human, primate, and dog cells The cell surface receptor for the RD114 env, RDR, is a neutral amino acid transporter [17] Northern blot analysis suggests that RDR is widely expressed in human tissues, including hematopoietic cells [18] It has been reported that transiently produced RD114-pseudotyped virus efficiently targets human CD34ϩ cells [19] and that RD114pseudotyped virus can be efficiently concentrated by ultracentrifugation [20] These two aspects of the RD114 envelope make it an attractive candidate for use in a stable retroviral packaging system A stable packaging cell line using MLV pseudotyped to RD114 has previously been described, a human-derived cell line, FLYRD18 [21] This packaging cell produces viral supernatants containing an unknown component that adversely affects the engraftment potential of human CD34ϩ cells [22] In this paper we describe a stable NIH3T3-based oncoretrovirus packaging cell line with an RD114 envelope that generates high-titer RD114pseudotyped virus that efficiently transduces human hematopoietic cells RESULTS RD114 Expression in Stable Retroviral Packaging Clones The GP101 cell line is a mouse NIH3T3 cell line stably transfected with a construct expressing the MLV gag and MOLECULAR THERAPY Vol 8, No 5, November 2003 Copyright © The American Society of Gene Therapy pol genes (Fig 1A) [3] We transfected GP101 cells with the RDF plasmid, an RD114 expression vector containing the RD114 env gene and a selectable marker, the phleomycin gene, expressed from the same mRNA and controlled by a FB29 Friend MLV LTR promoter (Fig 1B) [21] We analyzed 15 phleomycin-resistant clones by Northern blot to quantitate RD114 expression (Table 1) We saw two bands at approximately 2.9 kb and determined them to be the RNA expression products of the RDF plasmid based on the expected size of the RD114 gene mRNA from the RDF plasmid (data not shown) No bands were seen in the GP101 control RNA lane All clones including the GP101 control had a 1.4-kb band when probed with the G3PDH probe Two clones, 21 and 22, had very high RD114 envelope expression when normalized to G3PDH expression (Table 1) We used these high-expressing clones, 21 and 22, as well as a middle-range RD114-expressing clone, 13, to make viral producer clones and analyzed them for their ability to produce high-titer virus (Table 2) RD114 Viral Producers and Concentration of Viral Supernatants We transfected the RDF 13, 21, and 22 packaging clones with the retroviral vector p141␤ (Fig 1D) The p141␤ vector contains a human ␤-globin gene inserted in the reverse orientation and a neomycin-resistance gene (NeoR) controlled by a PGK promoter This previously described ␤-globin cassette has a 372-bp deletion in in- 805 ARTICLE doi:10.1016/S1525-0016(03)00263-6 blood [23] We also transfected RD114 packaging clones 21 and 22 with the SINPGKNeo vector (Fig 1C) The SINPGKNeo vector was made by deleting the ␤-globin gene and LCR elements from the p141␤ vector We isolated SINPGKNeo G418-resistant clones and p141␤ G418resistant clones and quantitated virus production by titration on HeLa cells The titers of individual producer clones are shown in Table We obtained the highest titers for both vectors using the RD114 packaging clone 21, SINPGKNeo clone with a titer of ϫ 105 viral particles/ml, and p141␤ clone with a titer of ϫ 104 viral particles/ml An amphotropic NeoR producer clone made from the GPϩenvAm12 packaging cell line [3] used as a control in these experiments had a titer of ϫ 103 on HeLa cells and ϫ 106 on NIH3T3 cells To verify full-length proviral integration of the p141␤ virus from producer clone 2, we did Southern blot analysis of DNA from a pool of transduced HeLa cells, which showed a single integration of the correct size proviral band (data not shown) We concentrated supernatants from our highest titer RD114 ␤-globin and NeoR producer clones by ultracentrifugation (see Materials and Methods) Titers of unconcentrated supernatants from the RD114 NeoR producer harvested over a period of days ranged from ϫ 104 to ϫ 105 viral particles/ml Fig 2A shows the viral titers of unconcentrated and concentrated supernatants from our RD114 NeoR producer The titers shown represent the averages of four experiments The titer was increased by approximately logs to ϫ 107 by ultracentrifugation The viral yield for these experiment varied within the TABLE 1: Analysis of RD114 packaging clone Clone designation RD114 RNA levelsa 0.33 0.34 0.49 0.70 10 1.65 13 1.94 15 0.40 16 1.80 17 3.40 18 2.30 21 8.90 22 12.10 23 0.46 25 1.18 26 0.70 Negative control—GP101 0.40 a Relative RD114 Northern blot signal normalized to G3PDH signal by dividing RD114 band density by G3PDH band density for each sample (see Materials and Methods) tron and several other modifications to increase the stability of proviral transfer The locus control elements HS2, HS3, and HS4 are upstream of the ␤-globin promoter [15] This vector has been used previously to transfer and express human ␤-globin in mouse bone marrow and TABLE 2: Titer analysis of three RDF packaging clones with two different retroviral vectors Titer (viral particles/ml)a Producer clone SINPGKNeoR retroviral vector p141␤ retroviral vector Packaging clone: 13 ϫ 101 1.4 ϫ 102 3 ϫ 101 3.3 ϫ 10 ND ϫ 10 ϫ 101 ND ND 10 21 22 0 ϫ 104 0 ϫ 102 ϫ 10 0 ϫ 102 13 21 ϫ 103 0 0 8.2 ϫ 103 6.8 ϫ 103 ϫ 10 ϫ 105 ϫ 103 1.5 ϫ 104 3.8 ϫ 10 7.1 ϫ 103 3.8 ϫ 102 1.2 ϫ 104 ϫ 10 1.1 ϫ 104 ϫ 104 4.2 ϫ 103 ϫ 10 ϫ 10 11 ϫ 103 2.6 ϫ 103 12 ϫ 10 ϫ 10 1 1.2 ϫ 104 ϫ 10 2.2 ϫ 104 ϫ 103 1.8 ϫ 104 3.1 ϫ 10 1.2 ϫ 104 ND, not done a Number of G418 HeLa clones/ml of viral supernatant 806 MOLECULAR THERAPY Vol 8, No 5, November 2003 Copyright © The American Society of Gene Therapy doi:10.1016/S1525-0016(03)00263-6 ARTICLE FIG Production and concentration of RD114 and amphotropic pseudotyped MLV NeoR virus Viral titers (viral particles per milliliter) were determined on HeLa cells (A) Concentration of RD114 MLV NeoR viral supernatants harvested over days Titers are the averages of two independent experiments (B) Concentration of amphotropic MLV NeoR supernatants over a 4-day culture period Titers are the averages of four independent experiments Diamonds represent viral titers of unconcentrated viral supernatants and squares represent viral titers of concentrated viral supernatants MOLECULAR THERAPY Vol 8, No 5, November 2003 Copyright © The American Society of Gene Therapy 807 ARTICLE range of 33 to 100% daily with an average yield of 60% Similarly, ultracentrifugation of RD114 ␤-globin supernatants increased titers from 1–9 ϫ 104 to 1–9 ϫ 106 (data not shown) In comparison, we also concentrated amphotropic virus Fig 2B shows the viral titers (the averages of two experiments) from unconcentrated and concentrated amphotropic NeoR supernatants The titer of unconcentrated virus was in the range of 1–2 ϫ 103 and was concentrated to 1–2 ϫ 105, again a 2-log increase in titer However, the titers are still lower than those of the RD114 producer The viral yield of amphotropic virus after concentration was similar to that of RD114-pseudotyped virus Analysis for Production of Infectious Wild-Type Virus We tested supernatants from pools of G418-resistant HeLa clones transduced with RDF21 SINPGKNeo supernatants for the ability to make replication-competent retrovirus; (RCR) To this, HeLa cells were infected with a 1:100 dilution of supernatant from the RDF21 SINPGKNeo producer, selected with G418, and then cultured for weeks Supernatants from these pooled G418-resistant HeLa clones were used to transduce fresh HeLa cells These infected cells were selected with G418 to detect the expression of the transferred SINPGKNeo vector No surviving G418-resistant cells were seen in two independent experiments using two different pools of transduced HeLa cells, indicating the absence of RCR Transduction of Human Cord Blood CD34؉ Cells To investigate the efficiency of transduction of human hematopoietic cells using our MLV-RD114 producer lines, we isolated CD34ϩ cells from human cord blood using the negative selection system from Stem Cell Technologies (Vancouver, BC) Cell samples were enriched to 46 – 62% CD34ϩ as measured by flow cytometry The progenitor colony potential (number of methylcellulose colonies per cells plated) was increased by 50- to 176-fold upon negative selection for an enriched CD34ϩ cell population, indicating that the mononuclear cell population had been enriched for a progenitor population We transduced aliquots of CD34ϩ cells with either unconcentrated or concentrated NeoR viral supernatants at varying multiplicities of infection (m.o.i.) (see Materials and Methods) The transduction efficiency was measured by scoring the number of G418-resistant colonies in methylcellulose assays and by PCR analysis for the presence of the NeoR gene in individual colonies The number of progenitor colonies grown without G418 varied in the range of 100 to 250 colonies per 103 cells for different samples Using unconcentrated NeoR viral supernatants at m.o.i of and 4, the transduction efficiency of CD34ϩ progenitor colonies based on PCR analysis for the NeoR gene in progenitor colonies was 10 and 20%, respectively (data not shown) We did not see significant numbers of G418-resistant colonies (Table 2) Using concentrated vi- 808 doi:10.1016/S1525-0016(03)00263-6 TABLE 3: Transduction efficiency of cord blood CD34ϩ cell progenitor colonies with RD114 concentrated and unconcentrated NeoR viral supernatants Experiment Unconcentrated virus Concentrated virus M.o.i.a % G418-resistant coloniesb (0/164) (0/196) 10 65 (110/170) 16 57 (119/208) 60 (118/197) 4 73 (95/129) 5 35 (87/250) 12 65 (182/280) 78 (217/277) 32 (70/213) 18 90 (177/191) 10 10 69 (86/123) 11 10 57 (77/135) a Multiplicity of infection; virus-to-cell ratio b No of colonies growing in G418 selection/No of colonies growing in no selection rus, the percentage of G418-resistant colonies from transduced cells ranged from 35 to 78% with an average of 62% (Table 3), a much greater efficiency than was achieved using unconcentrated RDF 21 Neo supernatants In the small range of m.o.i used for concentrated virus (4 to 16) there was no obvious increase in transduction efficiency with increased m.o.i We also picked colonies and analyzed them by PCR for the presence of the NeoR gene PCR analysis of colonies grown without G418 (% NeoR PCRpositive colonies) closely reflected the G418-resistance data (data not shown) All G418-resistant colonies with sufficient DNA content, as measured by PCR for the endogenous human ␤-globin gene, were PCR positive for the NeoR gene A previous report using supernatant from another RD114 stable producer cell line, FLYRD18, to transduce CD34ϩ cord blood cells showed that this supernatant altered the phenotype of these cells, causing a loss of the primitive CD34ϩCD38Ϫ cells in the population, and therefore adversely affected the NOD/SCID engraftment potential of these cells We measured the percentage of CD34ϩ cells and CD34ϩCD38Ϫ cells present in each sample at times pre- and posttransduction Fig 3A shows the FACS analysis of cells, preincubation and posttransduction with mock supernatant (media) or RDF Neo concentrated supernatants There was no difference in the percentage of CD34ϩ or CD34ϩCD38Ϫ cells present in mocktransduced cells compared to RDF NeoR-transduced cells (Fig 3B) MOLECULAR THERAPY Vol 8, No 5, November 2003 Copyright © The American Society of Gene Therapy doi:10.1016/S1525-0016(03)00263-6 ARTICLE FIG FACS analysis of CD34ϩ cell pre- and posttransduction (A) A representative FACS analysis for quantitation of the percentage of CD34ϩ and CD34ϩCD38Ϫ cells preincubation and post mock or RDF Neo transduction The percentage positive cells over unstained controls is indicated (B) Summary of the percentage positive CD34ϩ and CD34ϩCD38Ϫ cells pre- and postincubation The percentages are the averages of five experiments Error bars represent the standard deviations Cells were incubated for a total of 72 h for all five experiments Transduction of Sickle Cell CD34؉ Cells We also used peripheral blood as a source of CD34ϩ cells from sickle cell patients undergoing exchange transfusion by apheresis Using a negative selection system, we enriched mononuclear cells to 26 – 89% CD34ϩ We transduced our first sample (SS1), CD34ϩ cells, with either concentrated RD114 NeoR or p141 ␤-globin viral super- MOLECULAR THERAPY Vol 8, No 5, November 2003 Copyright © The American Society of Gene Therapy natants using an m.o.i of to following the same transduction protocol as for cord blood Fifty-one percent (163/317) of progenitor colonies from the Neo-transduced sickle cell CD34ϩ cells were G418 resistant NeoR PCR analysis also revealed that 44% (16/36) of colonies not selected with G418 contained the NeoR gene Using PCR primers that amplify the IVS2 region of the ␤-globin 809 ARTICLE doi:10.1016/S1525-0016(03)00263-6 TABLE 4: PCR analysis of sickle cell progenitor colonies from RD114 ␤-globin-transduced CD34ϩ cells Sample M.o.i.a % Transduced coloniesb % Transduced colonies grown in G418 Mock transduced NA 0/10 —c SS1 0/48 3/3 SS2 30 5/66 2/2 SS3 20 2/38 — SS4 24 3/39 2/2 SS5 29 1/44 — NA, not applicable a Multiplicity of infection b Number of colonies positive for transferred ␤-globin/number of DNA-positive colonies c No colonies or inadequate DNA in sample as revealed by PCR for endogenous ␤-globin sequences gene and, therefore, distinguish between the endogenous and transferred ␤-globin genes, we analyzed the colonies from the p141␤-transduced cells We detected no transferred ␤-globin gene in unselected colonies (Table 4) However, three G418-selected colonies contained the transferred ␤-globin gene (Fig 4, Table 4) To improve our transduction efficiency we repeated this experiment using higher m.o.i and were able to show an increase in transduction efficiency (Table 4) We were able to achieve up to 7.5% (5/66) ␤-globin gene transfer into sickle cell progenitor colonies in one experiment (Table 4) All G418-resistant colony DNA samples showing an endogenous ␤s-globin band by PCR also contained the transferred ␤-globin gene DISCUSSION Gene therapy into HSCs is an attractive treatment for inherited globin disorders Toward this goal, we and others have developed stable murine fibroblast-based oncoretroviral packaging cell lines that have proven to be safe and reliable in their viral production capacities [3,4,24] Using improved ␤-globin retroviral vectors, several studies have demonstrated significant expression of a transferred human ␤-globin in murine HSC using oncoretroviruses [23,25] Translation of these results into human hematopoietic cells has been difficult due to lowlevel expression of viral receptors on human HSC and the low titers of complex retroviral vectors Our goal in this study was to develop a stable oncoretroviral packaging system using a viral envelope that could overcome these barriers and efficiently transduce human hematopoietic cells using a potentially clinically relevant protocol for human gene therapy trials in the future Here we report the construction and characterization of a stable murine-based MLV-RD114 packaging line Previous studies using a human cell-based packaging cell line producing RD114-pseudotyped virus (FLYRD18) induced differentiation of the target human CD34ϩ cells [22] Using our RD114 packaging cell line, we were able to establish RD114 NeoR and human ␤-globin retroviral producer lines with reasonable titers (1 ϫ 105 and ϫ 104 viral particles/ml, respectively) as measured on a human cell line, HeLa With a comparable packaging cell line using an amphotropic receptor, the viral titer on HeLa cells with the same NeoR vector was ϫ 103 viral particles/ml The lower titer of this amphotropic NeoR viral producer is not due to lower viral production, but to lower infectivity of the human HeLa cells, as reflected by the higher titer measured on target NIH3T3 mouse cells In addition to the higher transduction efficiency of human cells, a great advantage of the RD114 viral supernatants from our stable RD114 ␤-globin and NeoR producers is that they could be efficiently concentrated with minimal loss of virus compared to the amphotropic NeoR producer More significantly, we show efficient gene transfer into human hematopoietic progenitor cells with a retroviral vector containing the NeoR gene marker By concentrating the viral supernatants by two logs, we were able to expose the human CD34ϩ cells to higher concentrations of the virus, increasing the m.o.i from with unconcentrated virus to 16 with concentrated virus, and show a concurrent increase in the transduction efficiency of the human progenitor colonies (Table 3) Interestingly, at similar m.o.i., the concentrated virus was more efficient than the unconcentrated virus This could simply reflect FIG PCR analysis of G418-resistant colonies from ␤-globin-transduced CD34ϩ cells isolated from peripheral blood of sickle cell patients The arrows indicate the expected bands, a 936-bp endogenous ␤-globin band and a 536-bp band representing the transferred ␤-globin gene Lane is ␾x174-HaeIII DNA size marker, lane is human bone marrow DNA, lane is p141␤ retroviral plasmid, lanes through 13 are DNA samples from p141␤-transduced methylcellulose colonies grown in G418 810 MOLECULAR THERAPY Vol 8, No 5, November 2003 Copyright © The American Society of Gene Therapy ARTICLE doi:10.1016/S1525-0016(03)00263-6 an increase in virus-to-cell contact with the decreased volume of viral supernatant used or the removal of substances inhibiting transduction in the concentration process Therefore, our stable RD114 producer has two improvements over the amphotropic producer: higher infectivity of human CD34ϩ cells and improved transduction efficiency due to concentration of virus Transient [10] or inducible [9] viral production systems are currently necessary for the production of VSV-Gpseudotyped virus because of the toxicity of this viral envelope protein and lentiviral proteins when pseudotyping with lentiviruses VSV-G-pseudotyped virus also provides the advantage of concentration by ultracentrifugation Stable cell lines have advantages over transient transfection systems in their ease of use, reproducibility of virus production, and facility for safety testing In these studies, we show high efficiency transduction of human hematopoietic progenitors with a single exposure to RD114-pseudotyped retroviral particles at m.o.i ranging from to 16 using a stable producer line Studies using VSV-G-pseudotyped MLV in an inducible system have required m.o.i of 50 to 200 to achieve efficient transduction of HSC [9] Gene transfer efficiencies of vectors containing the human ␤-globin have been achieved using VSV-G-pseudotyped lentiviral vectors in murine bone marrow transplant systems [26 –28] Expression levels of human ␤-globin using these vectors were high enough to show disease correction of ␤-thalassemia [29] and sickle cell disease transgenic mouse models [27,28] The success of these experiments is due in part to the ability to concentrate the VSV-G-pseudotyped lentiviral supernatants used in these studies to over ϫ 109 viral particles/ml, obtaining m.o.i of greater than 50 and, therefore, achieving multiple proviral integrations per cell The ability to obtain higher titer virus that can transduce human hematopoietic cells rather than mouse HSC is more challenging when using a complex retroviral vector such as the p141␤ vector containing the human ␤-globin gene with its regulatory elements We have demonstrated transfer of a human ␤-globin gene into CD34ϩ progenitors from sickle cell patients using a relatively low m.o.i with our RD114-pseudotyped virus One previous publication has shown transfer of a ␤-globin gene into hematopoietic cells from human fetal liver using a similar ␤-globin retroviral vector in a GALV-pseudotyped virus and a 5-day transduction protocol with multiple viral exposures [30] The authors demonstrated a transduction level of ϳ55% in various progenitor populations as measured by the GFP marker on the vector This is similar to the level we achieved with our RD114 NeoR virus using a 3-day transduction protocol with a single virus exposure Studies are under way using our stable RD114 packaging line to improve our transduction efficiency of human hematopoietic cells with the ␤-globin virus and to use the RD114 envelope in lentiviral gene transfer systems MOLECULAR THERAPY Vol 8, No 5, November 2003 Copyright © The American Society of Gene Therapy MATERIALS AND METHODS Construction and analysis of viral packaging and producer cell lines All NIH3T3 packaging cells were grown in Dulbecco’s modified Eagle’s medium (DME; Life Technologies, Rockville, MD) and 10% newborn calf serum HeLa cells were grown in DME and 10% fetal calf serum (FCS) All cells were grown at 37°C and 5% CO2 The GP101 MLV gag- and pol-containing cell line has been previously used to make stable amphotropic viral packager and producer cell lines used in human clinical trials [3,5,7] Two million GP101 cells were transfected with 10 ␮g of RDF plasmid (Fig 1B) using Lipofectamine 2000 following the manufacturer’s recommendations (Invitrogen, Carlsbad, CA) Cells were selected with ␮g/ml phleomycin in four 24-well plates for 20 days Individual clones were trypsinized, replated, and expanded for expression analysis RNA was made from RD114 packaging clones using Trizol reagent (Life Technologies) following the manufacturer’s instructions Five micrograms of RNA was run on a 1.2% agarose gel and blotted by standard procedures Blots were probed with a 32P-labeled 1-kb probe made by PCR amplification of the RDF plasmid, washed, and exposed to film Band density was quantitated by ImageQuant software The blot was stripped and reprobed using a G3PDH cDNA control probe to quantitate G3PDH mRNA as a sample loading control (Clontech, Palo Alto, CA) Three clones expressing high levels of RD114 mRNA (clones 13, 21, and 22) was selected for further experiments Three RD114 packaging clones were transfected by calcium phosphate precipitation with 10 ␮g of retroviral vector SINPGKNeo or p141␤ (Fig 1C and D) Cells were selected in medium containing 800 ␮g of G418 for 10 days and individual clones were trypsinized, replated, and expanded for titer analysis High-titer clones were identified by titration of retroviral supernatants on HeLa cells Southern blot analysis was performed on HeLa cells transduced with p141␤ retroviral supernatants using standard methods for DNA isolation and restriction enzyme digestion DNA was cut with SacI, which excises a 5.2-kb fragment that encompasses the full transferred proviral sequence The NeoR gene was labeled with 32P by random priming and used as a probe Production of concentrated viral supernatants RDF 21 SINPGKNeo or p141␤ producer cells were plated in 175-cm2 tissue culture flasks and grown in DME, 10% heat-inactivated FCS until semiconfluent Cultures were fed fresh medium the day before harvesting virus Viral supernatants were harvested daily and cultures were refed fresh medium for subsequent daily harvest of virus Supernatants filtered through a 0.45-␮m filter unit and virus was concentrated by spinning at 100,000g in a SW28 rotor for 90 at 4°C Viral pellets were resuspended overnight in 100 ␮l of Iscove’s modified Dulbecco’s medium (IMDM; Life Technologies) supplemented with 20% BIT serum substitute (Stem Cell Technologies) per tube, pooled, and titered on HeLa cells the following day Concentrated virus was stored at Ϫ80°C until use Viral supernatants were harvested from cell cultures for up to days depending on the stability of the stromal cultures For comparison, GPϩenvAm12 amphotropic NeoR supernatants were also concentrated in the same manner Isolation and transduction of human CD34؉ cells One hundredto150-ml samples from fresh umbilical cord blood were obtained Peripheral blood samples were obtained from sickle cell patients undergoing exchange apheresis for clinical indications Human blood samples were diluted 2:1 with phosphate-buffered saline and mononuclear cells separated by Ficoll density gradient centrifugation A CD34ϩ-enriched population was isolated using a negative selection system (Stem Cell Technologies) following the manufacturer’s recommendations To determine the percentage of CD34ϩ and CD34ϩCD38Ϫ cells, the cells were stained with anti-human CD34-PerCP and anti-human CD38-APC (Becton–Dickinson, San Jose, CA) and analyzed on a FACSCalibur (Becton–Dickinson) using Cell Quest software (Becton–Dickinson) The CD34ϩ-enriched population was preincubated for 24 to 48 h at 37°C and 5% CO2 on Retronectin-coated plates (Takara Shuzo Ltd., Otsu, Japan) in IMDM supplemented with 20% BIT serum substitute (Stem Cell Technologies) as well as the growth factors 10 ng/ml thrombopoietin, 10 ng/ml interleukin 6, and 100 ng/ml Flt3 ligand, all from Peprotech (Rocky 811 ARTICLE Hill, NJ), as well as 100 ng/ml stem cell factor (kindly provided by Amgen, Thousand Oaks, CA) and 10 ng/ml granulocyte-stimulating factor (Immunex, Seattle, WA) After a 24- to 48-h preincubation period, cells were pelleted and resuspended in unconcentrated viral supernatants Cells were exposed to two changes of viral supernatant over a 24-h period Alternatively, aliquots of concentrated virus were added directly to the cultures after the preincubation period Control cells were mock transduced using the same volume of medium alone Cells were collected and counted the following day One thousand cells were plated in methylcellulose (Stem Cell Technologies) with or without mg/ml G418 (Life Technologies) and scored 14 days later PCR analysis of methylcellulose colonies Methylcellulose colonies from plated CD34ϩ cells were also analyzed by PCR for the presence of the NeoR transgene or the ␤-globin transgene using specific primers described previously [23] Colonies were picked, washed in phosphate-buffered saline, and resuspended in 200 ␮l of InstaGene Matrix (Bio-Rad, Hercules, CA) Samples were incubated at 55°C for 30 and then boiled for The matrix was pelleted and 25 ␮l of the aqueous sample was added to the PCR Colonies were scored for the presence or absence of the transferred NeoR or ␤-globin gene only if there was an adequate DNA sample as assessed by the amplification of the endogenous ␤-globin gene by PCR [23] doi:10.1016/S1525-0016(03)00263-6 12 13 14 15 16 17 18 19 ACKNOWLEDGMENTS This research was sponsored by NIH Grant HL-59887 A.J.B is a fellow of the Cooley’s Anemia 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transduced transplantable human fetal liver and cord blood cells Blood 100: 1257–1264 MOLECULAR THERAPY Vol 8, No 5, November 2003 Copyright © The American Society of Gene Therapy ... cell line with an RD114 envelope that generates high-titer RD114pseudotyped virus that efficiently transduces human hematopoietic cells RESULTS RD114 Expression in Stable Retroviral Packaging. .. characterization of a stable murine- based MLV -RD114 packaging line Previous studies using a human cell -based packaging cell line producing RD114- pseudotyped virus (FLYRD18) induced differentiation... therapy into HSCs is an attractive treatment for inherited globin disorders Toward this goal, we and others have developed stable murine fibroblast -based oncoretroviral packaging cell lines that