In utero transfer and expression of exogenous genes in sheep

Experimental Hematology 28 (2000) 17–30

In utero transfer and expression of exogenous genes in sheep Nam D. Trana, Christopher D. Poradaa, Yi Zhaob, Graça Almeida-Poradaa, W. French Andersonb, and Esmail D. Zanjania b

a Department of Veterans Affairs Medical Center, University of Nevada, Reno, Nev., and Gene Therapy Laboratories, University of Southern California School of Medicine, Norris Cancer Center, Los Angeles, Calif., USA

(Received 4 August 1999; revised 7 September 1999; accepted 9 September 1999)

Objective. We have previously reported that directly injecting low-titer retroviral vector supernatant into pre-immune sheep fetuses resulted in the transfer and long-term expression of the bacterial NeoR gene within the hematopoietic system of these animals for over 5 years. In the present studies, we investigated whether using a higher titer vector would enable more efficient transduction and expression of the transgenes within the hematopoetic cells in sheep injected in utero. Materials and Methods. Sixteen pre-immune sheep fetuses were injected intraperitoneally with the G1nBgSvNa8.1 helper-free retroviral vector supernatant encoding the bacterial NeoR and LacZ genes (titer: 13107 cfu/mL). Results. Over the 2-year time course of these studies, the presence and expression of the NeoR and LacZ genes were demonstrated in 12 of the 14 animals evaluated by several immunological and biochemical methods. Seven of the 12 sheep examined by flow cytometric analysis contained $ 6% transduced peripheral blood lymphocytes. Vector distribution was widespread without any detectable pathology. Importantly, PCR analyses and breeding experiments demonstrated that the germ line was not altered. Conclusions. These studies confirmed that direct injection of an engineered retrovirus is a feasible means of safely delivering foreign genes into a developing fetus and thus achieving longterm expression of the transgenes within the recipient’s hematopoietic cells. Furthermore, expression of the NeoR gene from these studies was higher than that reported in our previous study in which a lower titer vector was used. © 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc. Keywords: In utero—Gene therapy—Gene transfer—Fetus—Sheep

Introduction Recently, we reported the successful transfer and long-term expression of the bacterial NeoR gene in sheep following direct injection of a low titer retroviral vector into preimmune fetuses [1]. Preimmune sheep fetuses were injected intraperitoneally with retroviral preparations (supernatant, producer cells, or irradiated producer cells) and analyzed for the efficiency of transduction as well as the expression of the exogenous genes. Eight of the sheep that were evaluated for over 5 years consistently demonstrated presence and expression of the bacterial NeoR gene as determined by polymerase chain reaction (PCR) and G418-resistance assays. Additionally, transduction of the long-term repopulating cells was confirmed when secondary recipients transplanted

Offprint requests to: Esmail D. Zanjani, Ph.D., V.A. Medical Center (151B), 1000 Locust St, Reno, NV 89520 USA; E-mail: [email protected]

with hematopoietic stem cells (HSC) from transduced primary sheep exhibited transgene activity [1]. PCR analysis revealed the presence of proviral DNA in all tissues including the reproductive organs without pathology. There was no evidence of germ line alteration as assessed by PCR analysis and breeding experiments [1]. Thus, our previous studies demonstrated that the direct injection of an engineered retrovirus is a safe and efficient method of delivering a functional exogenous gene into a developing fetus that allows successful long-term expression of the transgene in the hematopoietic system to be achieved without placing the germ line at detectable risk. In the present studies, we investigated whether direct injection of a higher titer retroviral vector in utero would enable higher transduction efficiency and expression of the transgenes to be achieved within the hematopoietic cells. To address this question, sixteen pre-immune sheep fetuses were injected intraperitoneally with the G1nBgSvNa8.1

0301-472X/00 $–see front matter. Copyright © 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(99)0 0 1 3 3 - 2

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N.D. Tran et al./Experimental Hematology 28 (2000) 17–30

helper-free retroviral vector supernatant encoding the bacterial NeoR and lacZ genes (titer: 13107 cfu/mL). All the experimental fetal sheep survived to term and were born healthy. Two died as the result of unavoidable accidents at the farm during the first month of life. Of the 14 sheep that were evaluated, all carried the proviral DNA as assessed by NeoR- and lacZ-specific PCR up to 16 months post-injection. Additionally, high levels of G418-resistant hematopoietic progenitors were consistently observed. Over the 2-year time course of these studies, proviral DNA and high levels of G418-resistant hematopoietic progenitors were consistently observed in 12 of the 14 experimental sheep. Moreover, expression of the transgenes was also confirmed by immunofluorescence, ELISA, and flow cytometric analysis. Flow cytometric analysis of peripheral blood lymphocytes demonstrated that $ 6% transduction efficiency was achieved in 7 of the 12 sheep evaluated at 28 months postinjection. These studies confirm our previous results that retroviral-mediated gene transfer by direct injection is a feasible means of safely delivering functional exogenous genes into a developing fetus and achieving long-term expression of the transgenes within the recipient’s hematopoietic cells. In addition, expression of the NeoR gene in the sheep from these studies was higher than that reported in our previous studies in which a lower titer vector was used. Hence, there appears to be a positive correlation between the titer of the vector employed and the level of transgene expression in this novel model.

Materials and methods In utero gene transfer protocol The G1nBgSvNa8.1 retrovirus (titer: 13107 cfu/mL) is a Moloney murine leukemia virus (MuLV)-based vector encoding the bacterial lacZ and NeoR genes (Genetic Therapy, Inc., Gaithersburg, MD). Transcription of the lacZ gene is driven by the native viral LTR while the NeoR gene is under control of an internal SV40 promoter. The retroviral supernatant is free of replication-competent helper virus as determined by an extended sarcoma1/leukemia(S1/L-) assay [2]. Retroviral supernatant (0.2-0.6 mL) was injected intraperitoneally into pre-immune fetal sheep in utero as described previously [1,3]. After birth, peripheral blood and bone marrow samples were collected at regular intervals for evaluation of transgene presence and expression. NeoR- and lacZ-specific polymerase chain reaction Five hundred nanograms of total genomic DNA was used for PCR analysis as described by Saiki et. al [4] with some modifications; 0.5 mM of NeoR-specific primers were used to amplify a 440-base pair fragment of the bacterial NeoR gene [1]. Similarly, 0.5 mM of lacZ-specific primers were used to amplify a 247-base pair fragment of the E. Coli lacZ gene [5]. PCR reagents were used as described previously [1,5]. Samples were then amplified for 35 cycles performed with the following parameters: 958C for 30 seconds, 658C or 588C for 30 seconds for NeoR or lacZ, respectively, and 728C for 60 seconds; 20 mL of each reaction was electrophoresed on a 2% agarose, Tris-Borate-EDTA gel.

Southern blotting of PCR products Southern blotting was performed as previously described by Sambrook et al. [6]. After electrophoresis, the gel was transferred under denaturing conditions (1M NaCl, 0.4N NaOH) to Gene Screen Plus membrane (DuPont, Boston, MA). The DNA on the membrane was cross-linked by short wave UV irradiation for 15 seconds. The membrane was then prehybridized at 558C for 1 hour in RapidHyb (Amersham Life Science, UK); 23106 cpm of endlabeled NeoR or lacZ probes were added per milliliter of RapidHyb; the membrane was then hybridized at 558C for 2 hours. After hybridization, the membrane was washed at 558C for four times at 20-minute intervals, under conditions of increasing stringency. After washing, the membrane was autoradiographed for 1–3 hours at 2758C with one Biomax intensifying screen (Eastman Kodak, Rochester, NY). Immunofluorescent detection of neomycin phosphotransferase and b-galactosidase Smears of sheep peripheral blood or cytospins of sheep bone marrow mononuclear cells were prepared on glass slides as described previously [1]. After blocking non-specific binding, slides were then incubated with polyclonal rabbit anti-neomycin phosphotransferase (59 Prime-39 Prime, Inc., Boulder, CO) and monoclonal mouse anti-b-galactosidase (Sigma Chemicals, St. Louis, MO). Following incubation with the primary antibodies, FITC-labeled anti-rabbit IgG and PE-labeled anti-mouse IgM were then added to detect NPT and b-galactosidase, respectively. Nuclei were counterstained with DAPI (Oncor, Gaithersburg, MD) for neomycin phosphotransferase (NPT) and b-galactosidase. The slides were viewed on a fluorescent microscope fitted with a dual filter cube allowing the simultaneous visualization of DAPI and FITC or PE (Olympus America, Melville, NY). ELISA for NPT ELISA was performed with a commercially available kit according to the manufacturer’s instruction (59 Prime-39 Prime, Inc.). Hematopoietic progenitor assays The presence/function of the NeoR gene in hematopoietic progenitors was evaluated by measuring the resistance of hematopoietic progenitor cells to the neomycin-like antibiotic G418 (Gibco BRL, Gaithersburg, MD); 33105 bone marrow mononuclear cells were cultured in plasma clot (CFU-E, BFU-E) or methylcellulose (CFUGM) as previously described [7]. Mononuclear cells were cultured in erythropoietin (0.5 IU/mL), sheep PHA-stimulated leukocyte conditioned medium (PHA-LCM) (5% vol/vol), and in the presence or absence of G418 (0–3 mg/mL). Fluorocytometric detection of b-galactosidase Detection of b-galactosidase activity was performed with the FluoReporter lacZ Flow Cytometry Kit (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions. Briefly, peripheral blood and bone marrow mononuclear cells were isolated using Ficoll-Hypaque density gradient (Sigma Chemicals), washed and resuspended in cold (48C) 300uM chloroquine at 107 cells/mL; 100 mL aliquots of cell suspensions were incubated at 378C for 20 minutes. After incubation, 100 mL of prewarmed (378C) 2mM working solution of fluorescein di-b-D-galactopyranoside (FDG) was rapidly added to each sample tube and incubated for 1 minute at 378C; 1.8 mL of ice-cold staining medium containing 1 mg/mL propidium iodide and 300 mM chloroquine was added to each tube to

N.D. Tran et al./Experimental Hematology 28 (2000) 17–30 Table 1. Experimental summary

Sheep#/sex 690/M 691/M 692/M 693/F 694/F 695/F 696/M 697/F 698/M 699/F 700/M 701/M 703/F 704/F 705/F 706/F

Amount of G1nBgSvNa8.1 injected

Outcome

0.5ml 0.5ml 0.5ml 0.5ml 0.2ml 0.2ml 0.6ml 0.5ml 0.5ml 0.3ml 0.3ml 0.3ml 0.5ml 0.5ml 0.5ml 0.5ml

Positive/alive Positive/alive Positive/alive Positive/sac*. Positive/alive Negative/sac. Positive/died§ Positive/alive Positive/alive Negative/sac. died after birth¶ died after birth¶ Positive/alive Positive/alive Positive/alive Positive/sac*

This table summarizes the experimental design and the outcome of these studies. All animals were born alive and healthy. Only animals that expressed both NPT and b-galactosidase at 28 months postinjection are marked positive. *Sacrificed for safety studies; §died as the result of tetanus; ¶died as the result of accident at the farm.

stop FDG loading. Samples were kept on ice until analysis. FDGstained cells of the same type from three control animals were used to set the background autofluorescence compensation. FACS analysis of peripheral blood to detect NPT and b-galactosidase expression Peripheral blood mononuclear cells (13106) from experimental and control sheep were transferred to flow cytometer tubes and pelleted at 3503g for 10 minutes. The pellets were resuspended in cold 0.1% sodium azide PBS (Sigma Chemicals). Cells were fixed in 2% paraformaldehyde and permeabilized with 0.05% Tween 20. Nonspecific binding was blocked with 10% normal goat serum (Sigma Chemicals), and the cells were then stained with either rabbit polyclonal anti-NPT (59 Prime-39 Prime, Inc.), or mouse monoclonal anti-b-galactosidase (Sigma Chemicals). Expression of NPT and b-galactosidase was detected with FITC-labeled mouse monoclonal antibody to rabbit IgG (Sigma Chemicals) and PElabeled goat monoclonal antibody to mouse IgM (Immunotech, France) and analyzed using the FACScan flow cytometer (Becton Dickinson Immunosystems, San Jose, CA). Peripheral blood mononuclear cells from normal control sheep stained with the same protocol were used as a negative control. Assay for b-galactosidase expression in situ Assays were performed as described previously by Sanes et al. [8] with modifications. Briefly, bone marrow mononuclear cells, selected with G418 (Gibco BRL) at a concentration of 1 mg/mL for 7 days, were placed on slides by cytospin. Slides were then fixed for 5 minutes at room temperature in 2% (v/v) formaldehyde, 0.05% (v/v) glutaraldehyde PBS solution (Sigma Chemicals). After washing with PBS, the cells were stained overnight at 378C with 5 mM K ferricyanide, 5 mM K ferrocyanide, 2 mM MgCl2 (Sigma Chemicals), and 1 mg/mL X-gal (diluted in dimethyl formamide to 2%) (Gibco BRL). The cells were counterstained with

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nuclear fast red, rinsed with H2O, and coverslipped using Permount mounting medium. Cells were then visualized using a light microscope fitted with a blue filter for increased contrast (Olympus America).

Results Experimental summary The overall outcome of the studies is summarized in Table 1. A total of 16 pre-immune sheep fetuses were injected intraperitoneally with 0.2–0.6 mL retroviral supernatant; all survived to term and were born alive and healthy. Experimental sheep #700 and #701 died within a few days after birth (attacked by coyotes) before samples could be collected for analyses. Bone marrow and blood samples from the remaining 14 experimental sheep were collected soon after birth and at intervals thereafter for NeoR- and lacZ-specific PCR analyses. Evidence of proviral DNA integration and transgene activity was obtained as early as 6 months postinjection (3 months after birth). At 12 months postinjection, bone marrow samples from all 14 experimental sheep exhibited proviral DNA integration as evaluated by NeoRand lacZ-specific PCR analyses shown in Figures 1A and 1B. PCR analysis performed on peripheral blood samples also demonstrated the presence of the transgenes (either NeoR or lacZ) in all 14 experimental sheep. Sheep #695 and #699 were sacrificed at 25 months postinjection because evidence of proviral DNA integration was no longer observed. At 28 months postinjection, proviral DNA was consistently detected in bone marrow and peripheral blood samples in all 12 of the remaining sheep determined by NeoR-specific PCR analysis and confirmed by lacZ-specific PCR analysis suggesting that successful transduction of primitive hematopoietic stem cells was achieved in these animals (Fig. 1C). Expression of the transgenes was also confirmed by immunofluoresence, ELISA, and flow cytometric analysis. At 29 months postinjection, sheep #706 was sacrificed for tissue distribution studies. Sheep #696 died of tetanus at 29 months postinjection; this loss was not believed to be caused by the retroviral supernatant or the surgery procedure. Additionally, experimental ewe #693 was bred with a control ram and sacrificed at 31 months postinjection. Samples from this animal and her two fetuses were collected for safety studies. CBC with differentials were also performed on all animals throughout the study period. Detection of b-galactosidase and neomycin phosphotransferase using immunofluorescent staining Expression of the lacZ gene in peripheral blood mononuclear cells was evaluated by immunofluorescence using commercially available monoclonal antibodies against b-galactosidase. Results presented in Fig. 2A demonstrate that b-galactosidase was expressed in all 14 experimental sheep

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N.D. Tran et al./Experimental Hematology 28 (2000) 17–30

Figure 1. Southern analyses demonstrate the persistent presence of proviral DNA in bone marrow and peripheral blood of in utero transduced sheep. Reagent represents all the constituents of PCR mixture except DNA. –Controls were DNA collected from either bone marrow or peripheral blood from normal control sheep. (A) Presence of NeoR gene in bone marrow (BM) and peripheral blood (PB) at 12 months postinjection. (B) Detection of lacZ gene in bone marrow and peripheral blood at 12 months postinjection. (C) Presence of lacZ gene in bone marrow and peripheral blood at 28 months postinjection.

examined at 15 months postinjection. The level of transgene expression was measured as the percentage of total numbers of cells that stained positive per 1000 white blood cells. Only cells with intense PE staining were scored as positive; no PE staining was observed in normal control sheep. Levels of expression in all 14 animals ranged between 0.40-

6.60% (average 5 2.44%). As demonstrated in Fig. 2B, blood smears from a control sheep showed no PE staining (blue stain is DAPI counterstain, which binds to all DNA) while the blood smear from sheep #690 showed intense PE staining over the DAPI counterstain suggesting a high level of b-galactosidase expression within these cells.

N.D. Tran et al./Experimental Hematology 28 (2000) 17–30

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Figure 2. Expression of exogenous genes in peripheral blood and bone marrow from in utero transduced sheep determined by immunofluorescence. (A) Detection of b-galactosidase in peripheral blood at 15 months postinjection, and neomycin phosphotransferase (NPT) in bone marrow at 28 months postinjection in all experimental sheep. (*) Samples were not tested. Shown here are the ratio of white blood cells per 1000 that expressed the transgene. (B) Representative photographs of blood smears from a normal control sheep and sheep #690. Blood smear from control sheep shows no cytoplasmic stain, except for DAPI, while smear from sheep #690 shows intense b-galactosidase PE-cytoplasmic stain over the DAPI counterstain.

Figure 2A also demonstrates the relative expression of NPT in the bone marrow of these animals at 28 months postinjection as determined by immunofluorescent staining. Levels of NPT expression ranging between 4.76% and 11% (average: 8.63%) were detected. As we previously reported [1], when a low titer vector supernatant was used, the average NPT expression was 0.97%. Thus a near eightfold in-

crease in long-term transgene expression was achieved in these studies using a higher titer vector supernatant. Detection of neomycin phosphotransferase by ELISA Neomycin phosphotransferase expression was also evaluated by ELISA on all experimental animals at 16 months postinjection. Figure 3 shows the expression of NPT in pg/

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N.D. Tran et al./Experimental Hematology 28 (2000) 17–30

Figure 3. Presence of NPT in bone marrow from in utero transduced sheep detected by ELISA. Shown are NPT measured in picograms/1.03106 mononuclear cells at 16 months postinjection after appropriate background normalization using three different negative control samples according to the manufacturer’s instructions.

1.03106 bone marrow mononuclear cells after appropriate background normalization according to manufacturer’s instructions. All animals expressed a significant amount of NPT ranging between 4.7 and 56.9 pg NPT/1.03106 (average 5 33.4 pg/1.03106 cells). In particular, NPT expression was $ 40 pg/1.03106 cells in 7 of the 14 experimental animals. This is a 2.5-fold increase in NPT expression when compared with animals treated with low titer vector supernatant [1]. Neomycin phosphotransferase activity studies An alternate method of studying the efficiency of gene transfer and the expression of the NeoR transgene in hematopoietic cells was to evaluate the ability of bone marrow hematopoietic progenitors to form clonogenic colonies in the presence of lethal concentrations of G418 in vitro. A lethal concentration of G418 for normal control sheep cell is 2 mg/mL as determined previously [1]. At this concentration, only a minimal number of CFU-GM colonies were observed in control animals. In comparison, bone marrow progenitors from 12 of the 14 experimental sheep, evaluated at 16 months postinjection, gave rise to a significant number of G418-resistant CFU-GM colonies (0%–35%) as shown in Fig. 4A, with 4 sheep exhibiting $ 30% resistant progenitors. It should be noted that bone marrow progenitors from sheep #694 and #697 did not give rise to resistant colonies although NeoR-specific PCR analysis (data not shown) and

NPT-specific ELISA analysis (Fig. 3) demonstrated the presence and expression of the NeoR gene. The reason for this discrepancy is unclear; however, variations in samples collected on different occasions for these analyses at 16 months postinjection may play a role in the discrepancy. To determine the role of the exogenous NeoR gene in the G418-resistant CFU-GM colonies, resistant colonies were removed from methylcellulose plates and subjected to NeoR-specific PCR analysis. Subsequently, Southern blot analysis demonstrated that all resistant colonies contained the bacterial NeoR gene as shown in Fig. 4B.

b-Galactosidase activity studies Because b-galactosidase possesses the ability to cleave the substrate, fluorescein di-b-D-galactopyranoside, into fluorescein and di-b-D-galactopyranoside, flow cytometry analysis was used to measure the intensity of fluorescein as an indicator of enzyme activity at the single cell level. In these studies, only viable lymphocytes were gated for initial analysis using propidium iodide. Figure 5A shows the percentage of mononuclear cells that exhibited b-galactosidase activity per 50,000 mononuclear cells at 22 months postinjection. All the animals studied exhibited b-galactosidase activity either in the bone marrow or in the peripheral blood; expression levels were between 0.13% and 2.92%. b-Galactosidase expression was also examined in situ using x-gal staining at 28 months post-treatment. b-Galactosidase

N.D. Tran et al./Experimental Hematology 28 (2000) 17–30

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Figure 4. Evidence of NPT activity in treated sheep. (A) CFU-GM colonies from bone marrow hematopoietic progenitors expressed as percentage of total colonies that were resistant to 2 mg/mL G418 at 16 months postinjection. (B) Detection of NeoR gene in DNA isolated from G418 resistant CFU-GM colonies by PCR. Negative control (CT) DNA was isolated from normal bone marrow colonies grown in the absence of G418 selection.

expression was evaluated in bone marrow mononuclear cells selected for 7 days at 1 mg/mL G418. Selected mononuclear cells were layered on slides by cytospin prior to x-gal staining. Figure 5B shows representative photographs of x-gal staining from a control sheep and experimental sheep #690. As demonstrated, the control sheep cells exhibited only nuclear fast red staining; however, significant x-gal blue stain was observed in cells obtained from sheep #690, indicating expression of b-galactosidase in this sheep. Hence, these studies suggested that the growth of these cells in medium with G418 was likely due to the expression of the transduced bacterial NeoR gene,

and that b-galactosidase was also being expressed simultaneously in the same population of hematopoietic progenitor cells. Although b-galactosidase was detected in the bone marrow of experimental sheep by the chromogenic method of detection (x-gal), the number of bone marrow cells that exhibited the enzyme was minimal when compared with flowcytometric analysis of b-galactosidase expression using fluorescein b-galactopyranoside. This observation was in agreement with previous studies [9–11] in which expression of b-galactosidase in bone marrow cells was shown to be below the level required for the chromogenic method of detection.

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N.D. Tran et al./Experimental Hematology 28 (2000) 17–30

Figure 5. Detection of b-galactosidase activity in blood and bone marrow of treated sheep. (A) Presence of b-galactosidase activity detected by fluorescence-activated cell sorting (FACS) in peripheral blood and bone marrow of experimental animals at 22 months postinjection shown as percentage of viable lymphocytes that exhibited enzyme activity per 50,000 mononuclear cells. (B) Representative photographs of x-gal staining of bone marrow mononuclear cells from a control and experimental sheep #690. Only cells with nuclear fast red stain were observed in control sheep whereas significant x-gal blue stain was observed in cells from sheep #690.

N.D. Tran et al./Experimental Hematology 28 (2000) 17–30 Table 2. FACS analysis of NPT and b-galactosidase expression in peripheral blood of in utero transduced sheep Total PB white blood cells Sheep # 690 691 692 693 694 696 697 698 703 704 705 706

Granulocytes/ monocytes

Lymphocytes

NPT

b-Gal

NPT

b-Gal

NPT

b-Gal

0.2 2.5 2.5 1.7 2.1 9.0 5.0 1.8 0.6 2.8 7.0 5.0

5.8 3.1 1.0 1.4 0.8 2.1 3.7 2.2 1.9 4.7 4.1 1.5

8.0 3.3 7.3 6.9 1.7 7.8 9.7 8.0 3.0 3.4 6.4 4.3

11.0 4.4 2.5 1.4 0 11.5 4.6 2.2 2.6 6.7 4.5 1.9

2.2 20.0 23.2 15.4 15.4 27.9 41.9 15.7 7.1 19.8 47.3 30.4

6.6 0.9 1.1 0.2 0 0 0.6 0 0 0 0 0

Data obtained from FACS analysis of peripheral blood white cells from in utero transduced sheep using antibodies to NPT and b-Galactosidase at 28 months postinjection are presented. Values represent the percentage of positive cells in each gated cell population after subtracting the values obtained with cells from age-matched control sheep using the same gates.

Multilineage expression of neomycin phosphotransferase and b-galactosidase The presence of proviral DNA and the detection of transgene expression in peripheral blood and bone marrow mononuclear cells for as long as 28 months post-treatment suggested that successful transduction of primitive hematopoietic stem cell subsets was achieved. This was also shown by multilineage expression of the NeoR and lacZ genes. FACS analyses for the presence of b-galactosidase and NPT in peripheral blood mononuclear cells were performed at 28 months postinjection to confirm long-term expression of the transgenes. Results presented in Table 2 demonstrate the multilineage expression of NPT in these experimental animals. After normalization with control samples, NPT was detected in the ungated whole blood population (0.2%– 9.0%), the lymphocytic population (1.7%–9.7%), and the granulocytic/monocytic population (2.2%–47.3%). Specifically, average NPT expression from 12 experimental animals was 3.40% in the ungated whole blood population, 6.04% in the lymphocytic population, and 22.42% in the granulocytic/monocytic population. Similarly, FACS analysis for b-galactosidase also demonstrated significant multilineage expression in all experimental animals. Specifically, b-galactosidase expression

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was between 0.8% and 5.8% in whole blood, 0% and 11.5% in the lymphocytic population, and 0% and 6.6% in the granulocytic/monocytic population (Table 2). Average b-galactosidase expression was 2.70% in the whole blood population, 4.43% in the lymphocytic population, and 0.66% in the granulocytic/monocytic population. Although the highest NPT expression was detected in the granulocytic/monocytic population, the highest b-galactosidase activity was observed in the lymphocytic population. The reason for this difference is unclear. Safety evaluations Possible cytotoxicity caused by the expression of the transgenes and the safety of the gene transfer procedure were also evaluated. Table 3 compares the peripheral blood values in the 12 experiment sheep with age-matched control animals at 16 months post-treatment. No significant differences in any of the parameters were seen. Evaluation of tissues from animals #695 and #699 (treated but found to be negative), sheep #706 and #696 (both positive), and sheep #693 (positive) at 25–31 months postinjection did not reveal any abnormalities. This despite the fact that, as was reported previously [1], proviral DNA was present in nearly all tissues: brain, spleen, liver, kidney, bladder, thymus, lung, stomach, intestine, skin, heart, peripheral blood, bone marrow, and ovary examined for lacZspecific PCR analysis (Fig. 6). The detection of proviral DNA in the ovary of ewe #706 raised the possibility that the germ line may have been altered. To investigate this possibility, ewe #693 was bred with a control ram and sacrificed at 60 days postbreeding for evaluation. Bone marrow, blood, and reproductive tissues were collected from the mother and twin fetuses A and B for analysis. As shown in Fig. 7A, lacZ-specific PCR analysis demonstrated that none of the samples from the two fetuses exhibited the proviral DNA, although proviral DNA was detected in blood, bone marrow, and ovary of the mother (sheep #693). Ejaculates were collected from all experimental rams (#690, 691, 692, and 698) on five different occasions and analyzed by lacZ-specific PCR and fluorescence in situ hybridization (FISH). A representative immunofluorescent sperm smear from experimental ram #698, shown in Fig. 7B, demonstrates that the whole ejaculate from ram #698 exhibited b-galactosidase activity. However, the transduced cells were not sperm cells. Phenotype analysis revealed that

Table 3. Circulating blood cell values

Control Experimental

Lymphocytes

Monocytes

Eosinophils

Basophils

Neutrophils

% Hematocrit

66.4 6 8.2 74.9 6 9.8

7.6 6 4.4 3.8 6 2.7

3.6 6 3.5 1.0 6 0.9

0.2 6 0.5 0.4 6 0.2

21.6 6 5.0 20.2 6 10.4

40.0 6 2.0 35.3 6 3.5

Each value represents mean 6 SD of results from 14 experimental and 3 age-matched control sheep at 16 months (i.e., 13 months after birth) post-treatment. The values were obtained by counting 100 cells.

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Figure 6. Distribution of proviral DNA in tissues of experimental sheep #706 at 29 months postinjection. LacZ-specific PCR analysis demonstrated the presence of proviral DNA in all tissues examined except for brain tissue. Negative control (CT) DNA was obtained from a normal control sheep bone marrow.

the b-galactosidase cell was of blood origin. Whole ejaculate from each animal was divided into three populations for analysis: whole ejaculate, purified sperm, and leftover ejaculate after purification using Percoll density gradient centrifugation as previously described [1]. Samples from each population were then analyzed by FISH and lacZ-specific PCR. FISH performed on these sperm populations indicated that proviral DNA did not integrate into the germ line (data not shown). As shown in Fig. 7C, proviral DNA was not detected in any of the purified sperm cell populations determined by lacZ-specific PCR analysis. Although proviral DNA was detected in the whole ejaculate from ram #698, this was expected because the sperm smear, shown in Fig. 7B, demonstrated the presence of transduced nonsperm cells.

Discussion The results presented here demonstrate that the direct injection of an engineered retrovirus into the peritoneal cavity of preimmune sheep fetuses is a safe and relatively efficient method of delivering functional exogenous genes into hematopoietic cells of these animals. This conclusion was supported by the detection of proviral DNA in the peripheral blood and bone marrow mononuclear cells, the presence of G418-resistant hematopoietic progenitor in the bone marrow, and the expression of the exogenous genes in hematopoietic cells detected by immunofluorescence, ELISA, and FACS analysis. As is shown, the expression of the transgenes was documented for at least 28 months postinjection suggesting that successful retroviral-mediated gene transfer into primitive hematopoietic cells appears to have occurred. Although b-galactosidase was consistently detected in the bone marrow and peripheral blood of all experimental sheep by immunofluorescence and flow cytometric analysis of b-galactosidase using fluorescein b-galactopyranoside (FDG), b-galactosidase expression was not consistently detected using the chromogenic method of detection (x-gal staining). Similar observations were also reported by other investigators [9–11], suggesting that x-gal staining is not sensitive enough to detect low level expression of b-galac-

tosidase in bone marrow and peripheral blood mononuclear cells. Flow cytometric analysis of b-galactosidase was found to be a more sensitive method of detection. Retroviral-mediated gene transfer was not limited to the hematopoietic cells. Cells from spleen, liver, kidney, bladder, thymus, lung, stomach, intestine, skin, heart, and ovary were found to contain proviral DNA. However, all tissues that contained the transgenes appeared to be histologically normal. Furthermore, hematocrits, white cell counts, and differential counts were within normal limits suggesting the absence of significant pathologic influence of the in utero transfer procedure. These findings suggest that replicationcompetent virus did not arise during the long period of observation in these animals. Although injected with the retroviral supernatant free of replication-competent helper virus as determined by the extended sarcoma1/leukemia2 (S1/L2) assay, the widespread distribution of proviral DNA in in utero transduced sheep raises the possibility that replication competent virus may arise during the course of in vivo expansion; however, this is not likely. We have previously reported [1], and also show here, that the presence of proviral DNA in these tissues was not associated with any pathology. In our previous studies, seven fetuses were injected with the Pat2.4/G1TkSvNa.90 and the Pa317/ G1NaSvAd.24 producer cells and analyzed after birth [1]. Even when injected with non-irradiated producer cells, no evidence of replication competent virus was detected in any of these animals for up to 6 years at last tested. The normal CBC/differential and persistence of transgene expression also indicate the absence of a cellular-or humoralmediated response to the proviral proteins. This is in contrast to previous in utero gene transfer attempts in older, immunocompetent sheep fetuses in which cellular-mediated responses against the viral encoded proteins and transduced cells resulted in transient, short-term transfer, and expression of the transgenes [12–16]. The long-term persistence and expression of the vector-encoded genes in our studies was likely due to the pre-immune status of the fetuses at the time of vector injection. During early fetal development, there is a window of opportunity, prior to thymic processing of mature

N.D. Tran et al./Experimental Hematology 28 (2000) 17–30

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Figure 7. Absence of proviral DNA in germ line from in utero transduced sheep. (A) LacZ-specific PCR analysis demonstrated the absence of proviral DNA in the progenies of in utero transduced ewe #693 although peripheral blood, bone marrow, and ovary from this ewe contained the proviral DNA. (B) Presence of contaminated nonsperm cells that expressed the viral encoded genes detected by b-galactosidase specific immunofluorescence on smears made from whole ejaculates. b-galactosidase was detected in nonsperm cells, shown in red (PE) over DAPI, in sperm smear from ram #698. (C) LacZ-specific PCR analysis demonstrated the absence of proviral DNA in all purified sperm samples from experimental sheep. Abbreviations: W.E. 5 whole ejaculates; L.E. 5 leftover ejaculates after Percoll density gradient. Control samples were obtained from an untreated age-matched normal ram.

lymphocytes, during which the fetus is tolerant of foreign antigens [17,18]. In addition, exposure of the fetus to antigens during this window of opportunity results in sustained tolerance, which could be permanent if antigens are consistently

maintained [19,20]. Ideally, retroviral-mediated gene transfer initiated during this window of opportunity would prevent any incidences of cellular-mediated immune response to both the vector-encoded proteins and the vector-containing cells.

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N.D. Tran et al./Experimental Hematology 28 (2000) 17–30

The pre-immune status of the fetus and the route of administration may also have circumvented the rapid in vivo inactivation of the murine retroviral vectors [21–23]. Characteristics of the developing fetus offer a unique opportunity to treat a wide variety of genetic diseases by gene therapy in utero. Carrying out gene transfer early in gestation would enable the delivery of the therapeutic gene before the genetic deficit has significantly compromised the patient. In addition, several features of the developing fetus make it an ideal target for HSC-directed gene therapy. The highly proliferative status of the fetus is one of these characteristics. Numerous studies have now provided evidence that a higher percentage of the HSC clones present within a fetus are actively cycling at any given point in time than would be in an adult counterpart [17,18]. For this reason, fetal HSC should be more amenable to gene transfer with the existing generation of murine retroviral vectors, which require cell division for transduction to occur. In this regard, the recent clinical studies performed by Kohn et al. [24] in which neonates with ADA deficiency were transplanted with gene-modified autologous umbilical cord blood HSC support the view that HSC from developmentally earlier sources may be better suited for retrovirus-based gene transfers. These trials have thus far demonstrated gene transfer and expression in significantly higher percentages of circulating leukocytes than previous trials performed on adult patients. Moreover, in vitro studies have demonstrated that more efficient gene transfer with retroviral vectors can occur in populations of hematopoietic progenitors from fetal donors when compared to adult cell populations [25,26]. Another aspect of fetal development that suggests a potential benefit for performing HSC gene therapy in utero is the expansion of the hematopoietic system during gestation. Conceivably, if transduction of even small numbers of primitive HSC could be accomplished early in gestation, expansion of transduced HSC could lead to significant transgene activity. In this regard, we have previously reported the successful transfer and long-term expression of bacterial NeoR gene in sheep following an in utero HSC transplantation/retroviral transduction protocol [25,26]. The procedure involved collecting peripheral blood from 110-day-old fetal sheep (term:145 days) by exchange transfusion with maternal blood, exposing the mononuclear cells to the N2 retroviral vector overnight, and then re-infusing these cells into the fetus. After birth, the NeoR sequence was detected in marrow and blood of many of these animals by PCR, and many of the animals contained G418-resistant hematopoietic progenitors of erythroid (CFU-E, BFU-E), myeloid (CFU-GM), and mix (CFU-Mix) phenotype, suggesting that primitive HSC may have been transduced. That HSC were transduced was further substantiated by the continued presence of G418resistant progenitors and PCR positivity in two animals that were observed for up to 43 and 59 months after birth [25]. This and the results of the direct-vector injection approach reported previously [1] and described here demonstrate that an

in utero approach to gene therapy could result in the transfer and long-term expression of the vector-encoded genes. For the in utero gene transfer approach to be effective, it must result in efficient transfer/expression of the transgene. In previous studies, we reported successful transfer and long-term expression of the exogenous gene following direct injection of a low titer (13104 cfu/mL) retroviral vector into pre-immune fetuses [1]. However, the transduction efficiency (0.97%, n 5 3) determined on peripheral blood smears by immunofluorescence was low. In contrast, when measured similarly, the higher titer vector (13107 cfu/mL) used here resulted in a transduction efficiency of 2.4% (n 5 14). The efficiency of gene transfer was even higher in all blood lineages when measured flow cytometrically. In sheep that were transduced with high titer vector, transduction efficiency detected in whole blood, lymphocytes, and monocytes/granulocytes was 3.40%, 6.04%, and 22.42%, respectively, whereas, transduction efficiency was 0.69%, 0.80%, and 3.42% in whole blood, lymphocytes, and monocytes/granulocytes, respectively, in sheep that were transduced with low titer vector. Similarly, ELISA analysis demonstrated a significant increase in the production of neomycin phosphotransferase in sheep that were transduced with high titer vector when compared to low titer vector, 33.4 pg/13106 cells vs. 13.3 pg/13106 cells, respectively. These results confirm the correlation between transduction efficiency and the titer of the vector used [27]. While analyzing the animals in these studies, we observed that the levels of gene transfer and expression in the bone marrow progenitors, as assessed by G418 resistance progenitor-derived colony assays, were significantly higher than those observed in blood. This is in agreement with our previous studies [1] and studies reported by others [24] where higher levels of transgene expression in the bone marrow progenitors were also observed. The difference in the transduction efficiency between bone marrow progenitors and mature blood cells is unclear. It is possible that this discrepancy may be due to the process of clonal succession where only a small percentage of the transduced HSC clones gives rise to mature hematopoietic cells at any given time in vivo as suggested by Lemischka and colleagues [28]; in vitro, in the presence of growth factors, more of the affected clones may form progenies that exhibit resistance to G418. Another important consideration relates to the wide distribution of the vector in nearly all tissues including the reproductive organs. We have analyzed the possibility of germ line alteration by evaluating newborn lambs following breeding experiments in which either one or both parents expressed in utero administered transgene activity. None of the five offspring exhibited proviral DNA. Similarly, although ejaculates were on occasion positive, PCR analysis of purified sperm cells isolated from ejaculates obtained from each of the four treated rams on five separate occasions failed to show the presence of proviral DNA in sperm cells. Similar results were reported by Ye et al. [29]. In a

N.D. Tran et al./Experimental Hematology 28 (2000) 17–30

carefully designed study, Ye et al. [29] evaluated 578 offspring of matings in which either one or both parent mice were injected with a high dose of an E1- and E4-deleted adenoviral vector. The dose of the vector was sufficient to affect 80% of the hepatocytes with low level dissemination to ovaries and testes in 94% of the animals. No evidence of germ line transmission was seen. Thus, available evidence suggests that in utero gene therapy by a direct vector injection protocol does not put the germ line at significant risk of being compromised. It is also interesting to note that although there was approximately a 1000-fold difference in titer between the viral supernatant used in these studies and our previous studies [1], the increase in transduction efficiency was on the order of two- to eightfold, suggesting that there are other factors that may influence gene transfer. In this regard, although a higher percentage of HSC clones present within a fetus are actively cycling than would be in an adult counterpart [17,18], HSC are quiescent in nature and only a relatively small fraction may be in active cell cycle at any given time [30]. Thus, the quiescent nature of HSC is most likely the major limiting factor in gene transfer since MLV-derived retroviral vector is unable to transduce nondividing cells. In addition, the level of expression of amphotropic retroviral receptors may play a role in the transduction efficiency in this large animal model. Orlic and colleagues [31] have demonstrated the mRNA levels of the amphotropic receptor were low in human CD341/CD381 cells and very low in human CD341/CD382 cells by reverse transcriptase analysis. Furthermore, attempts to up regulate the expression of retroviral receptor by culturing HSC with interleukin-3 (IL-3), interleukin-6 (IL-6), and stem cell factor (SCF) did not result in increased levels of viral receptor mRNA [31]. We are planning to determine whether transduction efficiency can be enhanced with multiple injections of retroviral supernatant. In theory, this approach should result in improved transduction efficiency since additional cohorts of hematopoietic stem cells are likely to be exposed to the vector at the times of vector administrations. These studies confirm that direct injection of an engineered retrovirus is a relatively safe and efficient means of successfully delivering therapeutic genes into hematopoietic cells of a developing fetus with long-term expression of the transgenes. Additionally, these studies demonstrate that a direct correlation exists between the titer of the vector employed and the level of expression of viral-encoded genes. The widespread distribution of the proviral DNA in these animals without apparent toxicity suggests that this in utero gene transfer approach may also be useful for the safe delivery of therapeutic genes to a variety of other tissues. Acknowledgments Supported by Grants No. HL40722, HL46566, HL39875, and DK51427 from the National Institutes of Health and by the Department of Veterans Affairs.

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