Lentiviral Vector Gene Transfer into Fetal Rhesus Monkeys (Macaca mulatta): Lung-Targeting Approaches

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Lentiviral Vector Gene Transfer into Fetal Rhesus Monkeys (Macaca mulatta): Lung-Targeting Approaches Alice F. Tarantal,1,2,* Chang I. Lee,1 Jason E. Ekert,1 Ruth McDonald,1,2 Donald B. Kohn,3 Charles G. Plopper,1 Scott S. Case,3,† and Bruce A. Bunnell4 1

California Regional Primate Research Center and 2Department of Pediatrics, University of California, Davis, California 95616-8542, USA 3 Division of Research Immunology/Bone Marrow Transplantation, Childrens Hospital Los Angeles, Los Angeles, California 90027, USA 4 Children’s Research Institute, Department of Pediatrics, The Ohio State University, Columbus, Ohio 43205, USA †Present address: Cell Genesys, Inc., Foster City, California 94404, USA *To whom correspondence and reprint requests should be addressed. Fax: (530) 752-2880. E-mail: [email protected].

We previously reported the efficiency of gene transfer in fetal monkeys using retroviral vectors and an intraperitoneal (IP) approach. Here, we explored intrapulmonary administration to determine whether gene transfer can be limited to the developing lung. The HIV-1-derived lentiviral vector (VSV-G pseudotyped; 1
107 infectious particles/fetus), using the enhanced green fluorescent protein (EGFP) as a reporter, was directly injected into fetal lung with ultrasound guidance (n = 4; 55 or 70 days gestation; term 165 ± 10 days). Fetuses were monitored sonographically, fetal/maternal blood samples collected during gestation, and four of four healthy newborns were delivered at term. All lung lobes were positive for the transgene (≤ 1%) when assessed by PCR, and transgene expression was observed by direct fluorescence microscopy and flow cytometry. The results of this study show the following: (1) successful gene transfer in fetal monkeys using an intrapulmonary approach; (2) less transduction of non-pulmonary tissues with gene transfer at 70 days gestation compared with 55 days gestation or use of an IP approach; (3) that the pulmonary epithelium was EGFP-positive by immunohistochemistry; and (4) no evidence of transplacental transport of vector sequences or antibody responses in the dams. The results of these investigations indicate the efficiency of fetal gene transfer by intrapulmonary delivery, and emphasize the importance of the fetal monkey as a preclinical model system for exploring in utero genetic treatment strategies for pulmonary disorders. Key words: fetal gene transfer, HIV-1-derived lentivirus, EGFP, rhesus monkey, lung

INTRODUCTION The primary goal of gene therapy is to achieve successful gene transfer and expression in a target cell to correct a genetic disorder or treat an infectious disease. The importance of genetic treatment for fetuses diagnosed with inherited diseases has been the subject of several reviews [1–3]. The rationale for treatment during this time is to prevent severe clinical disease by early intervention. Gene therapy for pulmonary disorders such as cystic fibrosis (CF) has been described [4–9]. For gene transfer strategies for the treatment of lung diseases such as CF to be successful, it is necessary that administration occur before irreversible destruction of the airway epithelium occurs. Infants born with CF can have normal lungs at birth, but lung inflammation can occur as early as 4 weeks after birth [10–12]. In addition, studies have shown that fetuses examined after prenatal diagnosis of CF have tracheal epithelium atrophy with cells devoid of cilia [13], suggesting that

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aberrations are present before clinically apparent disease. Thus, fetal gene transfer may provide the best opportunity for inserting therapeutic genes before the onset of lung damage, with the delivery of healthy newborns at term. Prior gene therapy studies for the treatment of pulmonary disorders have shown that gene expression is greater in the growing rather than the mature airway, both in magnitude and duration of expression [14,15]. In addition, results indicated that adults could not be boosted with a second administration of a viral vector, whereas limited persistence of gene expression was circumvented by additional administration in neonates [14]. Clinical trials have shown that mature airway epithelial cells are relatively resistant to gene transfer, that host immune responses determine the duration of transgene expression, and that transduction efficiency is low [16]. Therefore, the functional immaturity of the fetal immune system may also eliminate a barrier that has

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FIG. 1. The HIV-1-derived lentiviral vector. The secondgeneration lentivirus EGFP vector pHRCMV-EGFP was used as the recombinant genome for the lentivirus vector. The lentivirus vector expresses the EGFP reporter gene from an internal CMV-IE promoter. The packaging plasmid, pCMVR8.91, was used to express the HIV-1 Gag, Pol, Tat, and Rev proteins required to package the recombinant lentiviral vectors without the HIV1 Env or accessory proteins Vif, Vpr, Vpu, and Nef. The envelope expression plasmid, pMD.G, was used to express the VSV-G envelope protein.

significantly hampered the development of effective gene therapy strategies in adults. We chose the fetal rhesus monkey for these studies because the pulmonary system of this species is most similar to humans developmentally and anatomically [17]. The fetal monkey lung passes through the same stages of development at similar gestational time points as the human fetus, and during prenatal and postnatal life the human and nonhuman primate lungs are still developing [18]. Humans and other primates share a mixture of cell phenotypes not found in other species [19], and the overall pattern of tracheobronchial epithelial differentiation is similar in humans [20] and rhesus monkeys [21]. Although there are some differences in the number of generations of the airways and the number and size of the alveoli, the overall structure in the monkey lung is more similar to that in the human than any other mammalian species including mouse, rat, rabbit, sheep, or pig [17]. Because our prior gene transfer studies have shown that intraperitoneal (IP) administration of retroviral vector supernatant to early gestation fetal monkeys results in most organ systems being positive for the transgene [22], we explored whether direct intrapulmonary administration of lentiviral vector supernatant (Fig. 1) could restrict transduction to the lung lobes. These studies have shown that intrapulmonary gene transfer during the embryonic stage of development (55 days gestation; term 165 ± 10 days) results in efficient gene transfer to the pulmonary epithelium, although a large

number of non-pulmonary tissues were also positive for the transgene. In contrast, gene transfer in fetal primates during the early second trimester (70 days gestation; pseudoglandular stage) was shown to result in less transduction of non-pulmonary tissues, and consistent transgene expression in the epithelial cells of the airways.

RESULTS The overall experimental design (Fig. 2) and outcome for these studies are summarized in Table 1.

Fetal Growth and Development After gene transfer, sonographic measurements of the fetal head, abdomen, and limbs in addition to gross anatomical evaluations were assessed weekly, as previously described, and all measures were compared with normative growth curves for rhesus fetuses [23]. All fetuses remained healthy during the study period. All parameters evaluated throughout gestation did not reveal any significant differences when compared with historical control fetuses of comparable gestational ages [23] (data not shown). All other parameters assessed (amniotic fluid volume, placental, skeletal, and organ development) did not reveal any differences when comparing gene-transferred to non-transferred fetuses. All complete blood counts (CBCs) and clinical chemistry analyses indicated that all hematopoietic, renal, and hepatic parameters were within normal limits when compared with control rhesus monkeys of comparable age (data not shown). A B Tissue harvests were performed at birth (approximately 3 months post-gene transfer). Assessment of organ weights and body measures indicated all were within the normal range compared with controls of comparable age (body weight for gene-transferred animals, 428.0 ± 11.1 g, versus control, 457.3 ± 9.2 g). Gross evaluations of the lung lobes, diaphragm, heart, and thoracic cavity FIG. 2. Overall experimental design. (A) Fetuses (n = 4) were administered the HIV-1-derived revealed normal structures, and each of the lentiviral vector at 55 or 70 days gestation (term 165 ± 10 days). Fetal blood (fb) samples were collected at 100 and 140 days gestation via ultrasound guidance for assessments of transduc- lung lobes (right and left cranial, right and tion and gene expression (flow cytometry, PCR, hematopoietic progenitor assays), then deliv- left caudal, accessory) were divided into mulered by hysterotomy at term for necropsy (*). MB, maternal blood samples. (B) Sonogram (coro- tiple sections for whole wet mount fluoresnal section) of a fetal monkey at 70 days gestation. Note the fetal heart and liver. Arrow indicates cence microscopy, flow cytometry, PCR, histhe location of the injection at the time of transfer (left lung lobe). l, Lung. tology, and immunohistochemistry.

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TABLE 1: Experimental design and outcome Viral vector HIV-1/VSV-G (1
107 infectious particles/fetus)

Promoter

Transfer approach

Gestational age (n)

Fetal outcome

Maternal outcome

CMV–IE

pulmonary

55 days (2)

term delivery, no adverse effects

returned to colony, no adverse effects

70 days (2)

term delivery, no adverse effects

returned to colony, no adverse effects

VSV-G, vesicular stomatitis virus-glycoprotein; CMV–IE, cytomegalovirus-immediate early.

Analysis of Reporter Gene Expression and Transfer Efficiency Whole tissue mounts of sections of each of the lung lobes were assessed under direct fluorescence microscopy. Evidence of enhanced green fluorescent protein (EGFP)-positive cells was found within the lung sections of all animals and on the pleural surface in some cases for the fetuses transferred at the earlier time point (55 days gestation; Fig. 3). Flow cytometric analysis of cell suspensions of the lung lobes was also performed, and 0.5–1% EGFP-positive cells were found in animals necropsied at birth (data not shown). The tissue distribution of EGFP gene expression was also assessed by flow cytometry of whole blood and cell suspensions of thymus, liver, spleen, cerebral cortex, cerebellum, and bone marrow. EGFP-positive fluorescent cells were found in cell suspensions of spleen (≤ 0.3%) from fetuses transferred at 55 days gestation only (data not shown). All other samples were negative. To analyze the distribution and persistence of transduced cells over time, animals were necropsied at birth and samples from all collected tissues were processed for PCR. PCR for the EGFP transgene in all lung lobes (right and left cranial and caudal, accessory) indicated that each of the collected lung lobes were positive for the transgene (≤ 1%). PCR results also indicated a greater percentage of EGFP-positive specimens from non-pulmonary tissues with intrapulmonary gene transfer at 55 days gestation

compared with 70 days gestation (Fig. 4A). For example, intrapulmonary gene transfer at 70 days gestation showed no evidence of the EGFP transgene in the brain (cerebral cortex, cerebellum), thymus, spleen, liver, pancreas, kidneys, lymph nodes, gastrointestinal system, diaphragm, muscle, or gonads. Outcome at 55 days gestation was similar to results achieved after IP administration (Fig. 4B and Table 2). With IP administration, 0.001–1% of the total genomic DNA was positive for EGFP, with some specimens at 10% levels [22]. We also established hematopoietic progenitor assays from fetal blood samples collected prenatally (100 and 140 days gestation) and from fetal blood, marrow, and select tissues (thymus, liver, spleen) at necropsy. All collected progenitors were assessed by PCR. There was no evidence of gene transfer into erythroid or myeloid progenitors with intrapulmonary gene transfer at either gestational time point (Table 2). These findings differed from results achieved with IP administration, as previously reported [22]. In this prior study, analysis of hematopoietic progenitor colonies from animals that were administered the HIV-1-derived lentivirus vector preparations demonstrated an average of 25% positive for EGFP. Morphology and Immunohistochemistry The cellular organization of the airways and parenchyma was assessed histologically in all lung lobes collected from

FIG. 3. Whole wet mount lung sections from a term fetus transferred at 55 days gestation and assessed by fluorescence microscopy. Note fluorescent cells within the lung (left) and on the pleural surface (right). Images were obtained within 3 h of tissue harvest.

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FIG. 4. EGFP PCR results after intrapulmonary gene transfer with the lentiviral vector at 55 versus 70 days gestation. (A) Collection of specimens from term animals transferred at 55 days gestation indicated multiple tissues, including all of the lung lobes, were positive for the transgene. In comparison, intrapulmonary gene transfer at 70 days gestation resulted in less transduction of non-pulmonary tissues. These findings differed when compared with outcome with IP administration with the same vector system at a similar titer (B). All specimens were positive for -actin (data not shown).

A

airways (Figs. 5A and 5B). This was a consistent finding in all lung lobes in all animals (4 of 4). Fetuses transferred at 55 days gestation also showed evidence of EGFP-positive cells in other organs and tissues including the spleen (red pulp), pancreas (acinar portion), muscle (myotubules), lymph nodes, and colon (mucosa) (Fig. 5C). There was no evidence of gene transfer into the germ cells of any of the fetuses evaluated, but further studies will be required to adequately address this issue.

B

all animals. There was no difference in the overall growth and size of the lung lobes, no evidence of inflammation, and the pseudostratified epithelium was unremarkable compared with specimens from control animals of comparable age (data not shown). The alveolar spaces were all relatively uniform and comparable in size compared with similar aged controls. Immunohistochemical assessments were carried out on sections of all collected lung lobes. Results revealed EGFP-positive cells within the surface epithelium of the

Antibody ELISA The generation of anti-EGFP and antilentiviral vector antibody responses were assessed in plasma samples collected from fetuses during gestation (100 and 140 days gestation) and at term by purified recombinant EGFP and concentrated empty R8.91 VSV (lenti packaging) serum-free preparations using indirect ELISA. There was no evidence of antibodies generated to EGFP or the vector construct in any of the animals assessed either during gestation or at term. Maternal Assessments All of the dams were sequentially monitored by evaluating CBCs, clinical chemistry panels, daily assessment of

TABLE 2: Results of EGFP PCR after fetal gene transfer using the HIV-1-derived lentiviral vector Route (N)

Brain Thymus Spleen

Liver

Pancr

Adrenals

Kidneys

Heart

Lung

LNs

GI

+

+

+

+

+

+

+

+

Muscle Gonads

HP

intrapulmonary 55 days (2)

+

+

+

+

+

-

70 days (2)

-

-

-

-

-

+

-

+

+

-

-

-

-

-

intraperitoneala (4) +

+

+

+

+

+

+

+

+

+

+

+

+

+

EGFP, enhanced green fluorescent protein; LN, lymph nodes (axillary, inguinal, mesenteric); GI tract, gastrointestinal tract (stomach, duodenum, ileum, jejunum, colon); HP, hematopoietic progenitors (erythroid, myeloid); + , positive by PCR; – , negative by PCR. Sensitivity of the EGFP PCR assay is 1 positive cell in 1
106 total cells. These samples were -actin assessed to confirm the presence of DNA (see text); a[22].

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A

FIG. 5. Immunohistochemical assessments. (A) Mid-level bronchus from rhesus monkey at term after intrapulmonary gene transfer using the lentiviral vector at 70 days gestation. The EGFP transgene observed within the surface epithelium (white arrow) (10
; frozen sections). (B) Comparison of specimens collected at 55 and 70 days gestation indicates a similar outcome. Negative control shown at right. (C) Other tissues collected at necropsy from a fetus transferred at 55 days gestation indicates that the colon and skeletal muscle were positive for the transgene.

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B

C

health, and monthly body weights. All maternal evaluations were within normal limits, and there was no evidence of adverse effects up through one year postdelivery of offspring (maximum time assessed). There was also no evidence of transplacental transfer of vector sequences in any of the dams evaluated. Plasma samples assessed at all of the collected time points (six time points per dam) for the generation of antibodies to EGFP or the vector construct were all negative.

DISCUSSION Lung development is a multi-event process that occurs both prenatally and postnatally, and consists of three general features: overall growth, branching morphogenesis, and cellular differentiation [18]. Fetal lung development occurs in four stages (embryonic, pseudoglandular, canalicular, saccular) with each stage occurring at a defined period of gestation. The timing of these stages is dependent on the species and length of gestation, with human and nonhuman primates more similar than rodents, rabbits, sheep, or pigs [18]. We chose to focus our intrapulmonary gene transfer approach on the embryonic and pseudoglandular stages because of the developmental events that occur at these time periods. In addition, our prior studies have suggested the level of immune system development during these stages would not interfere with gene transfer efficiency [24,25]. The studies described here indicate that direct intrapulmonary gene transfer in the late first trimester (terminal period of the embryonic stage) results in transduction of the pulmonary epithelium and a large

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number of non-pulmonary tissues. In contrast, gene transfer at 70 days gestation (latter period of the pseudoglandular stage) results in a more restricted transduction of tissues, with transduction of epithelial cells of the airways in all of the lung lobes. In all cases, there was no evidence of adverse effects or antibody responses as evidenced by histologic evaluation and indirect ELISA. The embryonic stage of lung development is characterized by the budding of the tracheobronchial tree from the primitive foregut, and includes the formation of the largest conducting airways by branching into the surrounding mesenchyme [18]. With further development, during the pseudoglandular stage, continued branching and budding of the bronchial tree is observed, until all of the airway generations characteristic of conducting airways in the adult have been formed down to the terminal bronchioles. The definition of this stage of lung development is derived from the fact that all of the tubules are lined by cuboidal- to columnar-shaped epithelial cells containing large amounts of glycogen, which spread into the surrounding mesenchyme. The vascular tree is absent from the growth of the lung up through these stages of development, which may explain the lack of transduction of hematopoietic cells. The difference in outcome when comparing gene transfer at the end of the embryonic stage of

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lung development (55 days gestation) with 70 days gestation may be explained by the difference in the size of the fetus at these two time periods. At 55 days gestation, the fetus is approximately 40 mm in length, and the body weight is approximately 4 g. By 70 days gestation, the fetus weighs approximately 35 g, and the lung lobes are of a considerably greater size. The diaphragm is completely formed by the earliest time of transfer performed in this study, therefore any viral vector supernatant that may have entered the thoracic cavity at the time of transfer would not have ready access to the abdominal organs. Based on our current findings, the data suggest that intrapulmonary gene transfer is more efficient at 70 days gestation for several reasons: (1) the transduction of nonpulmonary tissues is limited; (2) early pulmonary cell populations can be targeted; (3) fetal immune responses can be avoided; and (4) consistent expression of the transgene throughout all of the airways can be obtained. Uniformity of transgene expression within the lung lobes is most likely due to the fact that the fetal lungs are expanded with fluid rather than air at the time of gene transfer. Fluid movement throughout the airways could also account for the transduction of cells lining the airways in multiple lung lobes in each of the animals. Although direct injection of viral vector preparations into the amniotic fluid may seem a reasonable approach because the fetus swallows amniotic fluid, we did not include administration by this route for several reasons. First, amniotic fluid is a complex mixture of high and low molecular weight components that may inhibit or block the transduction of fetal cells [26]. Amniotic fluid has also been shown to have an impact on the transduction efficiency of viral vector systems when assessed in vitro [27–29] and in vivo [30]. Second, although the fetus swallows amniotic fluid during gestation, the injection of viral vector supernatant directly into the amniotic fluid would result in a large dilution of vector concentration, and a highly variable “dose” received by each of the treated fetuses. The dilution of vector concentration and exposure of other non-pulmonary tissues such as the skin and gastrointestinal system provide the greatest rationale against vector delivery by the intra-amniotic approach. Many gene delivery vectors use viral promoters such as the cytomegalovirus-immediate early (CMV-IE) promoter because this promoter can drive transgene expression in many cell types. Studies focusing on pulmonary gene transfer have used CMV-IE for this reason because it has yet to be resolved which of the 40 cell types in the respiratory tract needs to be targeted for each of the pulmonary disorders that may benefit from genetic therapy [5]. However, our prior fetal gene transfer studies [22] and many studies by others [31,32] have provided evidence of transgene silencing with this promoter. In addition, the development of pulmonary gene transfer strategies may require selective targeting of transgene expression to

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specific cells of the developing lung to ultimately be curative. Lung-specific promoters such as surfactant proteinC (SP-C; targets type II cells) or CC10 (targets Clara cells) are of interest for fetal gene transfer because SP-C mRNAs appear at early human fetal gestational ages in association with the epithelium where airways branch, and SP-C mRNA and proSP-C are present in distal lung epithelial cells before differentiation into type II cells occurs [33,34]. Whitsett et al. [35] used the gene CFTR under the direction of the SP-C and CC10 promoters in transgenic mice and evaluated the effects of expression in utero. The use of SP-C and CC10 promoters in these studies ensured CFTR expression in more differentiated cell types because these proteins become specific markers of their respective cell types (type II and Clara cell) as they differentiate. These studies show that direct intrapulmonary gene transfer is an efficient method for the transduction of the pulmonary epithelium in developing nonhuman primates, with no evidence of adverse effects in the fetus or dam. Although fetal gene transfer provides the opportunity to eliminate the pathology associated with life-threatening pulmonary conditions, there are important safety concerns which must be rigorously explored before considering its use in humans. For example, although we evaluated the organization of the airways, parenchymal volume, alveolar spaces, and overall lung growth, all of which were found to be normal, it is essential that lung function be assessed in postnatal animals in future studies. Because of developmental, anatomical, and physiological similarities, the fetal rhesus monkey is an ideal primate model system for these studies, and will continue to provide essential information on the safety and efficiency of novel fetal treatment strategies for future application in humans.

MATERIALS

AND

METHODS

Animals. All animal procedures conformed to the requirements of the Animal Welfare Act and protocols were approved before implementation by the Institutional Animal Use and Care Administrative Advisory Committee (AUCAAC) at the University of California at Davis. Normally cycling, adult female rhesus monkeys (Macaca mulatta) (n = 4) with a history of prior pregnancy were bred and identified as pregnant, using established methods [23]. Pregnancy in the rhesus monkey is divided into trimesters by 55-day increments with 0–55 days gestation representing the first trimester, 56–110 days gestation representing the second trimester, and 111–165 days gestation, the third trimester (term 165 ± 10 days) [36]. Activities related to animal care (diet, housing) and screening animals for endogenous retroviruses (SRV, STLV) before assignment to the study were performed as per standard California Regional Primate Research Center (CRPRC) operating procedures. Lentiviral vector preparation. The second generation HIV-1-derived lentivirus vector, pHR’CMV-EGFP, was constructed as described [37] (Fig. 1). This lentivirus vector expresses the EGFP reporter gene from an internal CMV-IE promoter. The packaging plasmid, pCMVR8.91, was used to express the HIV-1 Gag, Pol, Tat, and Rev proteins required to package the recombinant lentiviral vector without the HIV-1 Env or accessory proteins Vif, Vpr, Vpu, and Nef [38]. The envelope expression plasmid, pMD.G, was used to express the vesicular stomatitis virus-glycoprotein (VSV-G) envelope protein [37]. The recombinant VSV-G pseudotyped lentiviral vector was produced by transient three-plasmid transfection of 293T cells, as described [22].

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Recombinant lentiviral vector titer analysis by EGFP gene expression. Infectious titers were determined by limiting dilution analysis on 293 cells (ATCC CRL-1573). The 293 cells were plated at 1
105 cells/well in a sixwell cell culture plate (Corning, Miami, FL) in D-10, and placed in a 37C incubator for 12 h to adhere. Cells were transduced with serial dilutions (1
10–3 to 1
10–5) of lentiviral vector supernatant and analyzed by flow cytometry for EGFP gene expression 48 h after transduction. Titers were calculated using the following formula: (number of 293 cells at the time of vector addition) (% EGFP+) / 100 / (1 / dilution) = number of infectious units (iu)/ml of medium. The titers after concentration of the VSV-G pseudotyped lentivirus vector preparations were 5–10
108 iu/ml. The lentivirus vector supernatants were analyzed for the presence of replication competent lentivirus (RCL) by infection of phytohemmaglutinin (PHA)stimulated human peripheral blood mononuclear cells (PBMC), followed by culture for 2 weeks and then assay of culture medium for the HIV p24 Gag antigen by ELISA (Beckman Coulter, Fullerton, CA). The vector preparation used in these studies did not contain RCL.

skin, muscle, bone, and bone marrow. All lung lobes (right and left cranial and caudal, accessory) were collected and divided into multiple sections for whole wet mount fluorescence microscopy, flow cytometry, PCR, histology, and immunohistochemistry. The placenta, membranes, umbilical cord, and decidua were also collected and assessed. Representative sections of all collected specimens were quick frozen over liquid nitrogen for PCR, and fresh cell suspensions prepared from select tissues (individual lung lobes, thymus, liver, spleen, cerebral cortex, cerebellum) for flow cytometry, using standard techniques. Sections of each lung lobe were fixed in 10% buffered formalin for at least 24 h for routine histology and immunohistochemical assays; fixed for 1 h in 1% paraformaldehyde then placed in PBS with fungizone for direct fluorescence microscopy; and snap frozen at the time of necropsy after embedding in OCT. Representative sections of all tissues preserved in formalin were embedded and sectioned at 5–6 m then stained with hematoxylin and eosin (H&E) for routine histology. Specimens from animals of comparable age without any interventions (controls) were similarly processed and analyzed in parallel.

Lentiviral vector administration and fetal monitoring. All pregnancies were sonographically assessed to confirm normal growth and development before gene transfer [23]. The dams were administered ketamine hydrochloride (10 mg/kg) for these and subsequent ultrasound examinations. On the day of gene transfer (either 55 or 70 days gestation; Fig. 2), immobilized dams were aseptically prepared for transabdominal ultrasound-guided fetal gene transfer. A total volume of 100 l of the lentiviral vector supernatant was injected into the parenchyma of the right or left lung lobes using a 25 gauge
3 inch spinal needle attached to a 1 ml tuberculin syringe (n = 4; Fig. 2B and Table 1). Post-gene transfer, sonographic measurements of the fetal head (biparietal and occipitofrontal diameters, area and circumference), abdomen (area and circumference), and limbs (humerus and femur lengths), in addition to gross anatomical evaluations (axial and appendicular skeleton, viscera, membranes, placenta, amniotic fluid) were assessed weekly, as described, and all measures were compared with normative growth curves for rhesus fetuses [23].

Hematopoietic progenitor assays. Progenitor assays were established, as described [42]. Fetal blood samples were collected in utero, and blood, marrow, and select tissues (thymus, liver, spleen) were also collected for assay at necropsy. Progenitor cell assays were established using 0.5
105 nucleated blood cells/plate and 0.2
105 nucleated bone marrow (or tissues) cells/plate in 1 ml Methocult GF+ H4435 (StemCell Technologies, Inc., Vancouver, Canada), and plated in duplicate or triplicate. Evaluation after a standard incubation period (10 days) included total colony counts, and quantitation of individual erythroid and myeloid progenitors (burst forming unit-erythroid, colony forming unit-erythroid (CFU-E), CFU-granulocyte macrophage (CFU-GM), CFU-Mix). Individual erythroid and myeloid colonies were collected and processed using routine techniques and stored at –20C until analyzed by PCR.

Fetal/maternal assessment and sample collection. Fetal blood samples (1–2 ml) were collected via ultrasound-guidance using standard techniques [39,40] at 100 and 140 days gestation (second and third trimesters) and at tissue harvest (150 days gestation) for CBCs, to assess transduction and gene expression (flow cytometry, PCR, hematopoietic progenitor assays), and antibody responses. Blood samples were also collected (10 ml) from a peripheral vessel from the dams at 20 days gestation, before gene transfer, 1 week post-gene transfer, then at 100, 120, and 140 days gestation, and at birth for CBCs, clinical chemistry panels, and to determine whether transplacental transport of vector sequences or antibody production to the vector construct occurred. Maternal health was monitored daily and body weights were assessed monthly. Hematology and clinical chemistry. CBCs were performed on fetal and maternal blood samples (0.25 ml). Collected samples were placed directly into microtainer tubes with ethylene diaminetetraacetic acid (EDTA, Bectin, Dickinson, and Co., Rutherford, NJ), and evaluated as described [40]. Term bone marrow and liver smears were also evaluated at necropsy for the determination of the relative percentages of immature and mature cellular components by direct examination of stained bone marrow and liver. All smears were stained with Wright-Giemsa for morphologic evaluations. Clinical chemistry analyses were also performed, as described [41]. Parameters assessed included sodium, potassium, chloride, creatinine, aspartate aminotransferase, alanine aminotransferase, lactic dehydrogenase, alkaline phosphatase, bilirubin, blood urea nitrogen, glucose, cholesterol, phosphorus, calcium, albumin, total protein, CO2 content, creatinine phosphokinase, -glutamyl transferase, and triglycerides. Tissue harvest/necropsy. Complete tissue harvests were performed at term using standard techniques [41]. Total body weights and measures (hand, foot, humerus, and femur lengths; biparietal and occipitofrontal diameters; head, arm, and chest circumferences; crown-rump lengths) were assessed, then all organs were removed and weighed including the brain (cortex, cerebellum), thymus, spleen, liver, lymph nodes (axillary, inguinal, mesenteric), pancreas, right and left adrenals, right and left kidneys, right and left gonads, stomach, duodenum, jejunum, ileum, colon, heart, diaphragm, thoracic wall,

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Flow cytometry. Fetal blood, marrow, and cell suspensions of select tissues were collected and assessed by flow cytometry (lung, thymus, liver, spleen, cerebral cortex, cerebellum). Tissues were prepared by manual dissociation, then filtered (Falcon cell strainer). Samples were assessed concurrently with specimens from non-transferred animals of comparable age (negative controls). Specimens were prepared for flow cytometry using the Coulter Q-Prep. A FACS Calibur (Becton-Dickinson, San Jose, CA) was used for these studies. EGFP fluorescence was collected in the PMT designated FL1, and 50,000 events were assessed per sample. Analysis was performed using CELLQuest software. DNA isolation and PCR analysis. PCR was performed on blood, bone marrow, and hematopoietic progenitors collected after growth in culture, and all tissues collected at necropsy. Genomic DNA was isolated (5
105 to 1
106 nucleated cells) using the Gentra System DNA isolation kit (Gentra, Minneapolis, MN), as recommended by the manufacturer. DNA was isolated from hematopoietic progenitor cell colonies by heating in 50 l of a 100 g/ml proteinase K solution at 65C for 1 h followed by 95C for 10 min to inactivate the enzyme. Genomic DNA was amplified (300 g for tissues and blood and 10 l of progenitor DNA solution), as reported [22]. PCR was performed using AmpliTaq Gold (Perkin Elmer, Norwalk, CT) for 30 cycles (-actin primers) or 39 cycles (EGFP primers). The sensitivity of the EGFP PCR assay is 1 positive cell in 1
106 total cells. -Actin was assessed to confirm the presence of DNA in each of the samples evaluated. To quantitate the percentage of transduced cells in tissue and blood samples, a standard curve was generated as reported [22], and results were normalized to -actin to calculate approximate percentages. Immunohistochemistry. OCT-embedded frozen tissue sections (6 m) were mounted on glass slides, air-dried for 4 h, then fixed in cold methanol and air-dried in preparation for immunohistochemistry. We used 1% bovine serum albumen (BSA) to block nonspecific binding. A 1:100 diluted EGFP polyclonal rabbit antibody (Clontech) was applied and incubated at room temperature overnight. Tissue sections were washed with PBS and incubated with fluorescent goat anti-rabbit antibody (Alexa 568) for 30 min, then washed with PBS and examined microscopically. ELISA antibody titers. Viral-specific IgG antibodies were quantified from maternal and fetal plasma, as previously reported, using indirect ELISA

MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy

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[24,43]. The anti-EGFP and anti-vector antibody responses were assessed by purified recombinant EGFP and concentrated empty R8.91 VSV (lenti packaging) serum-free preparations. Briefly, after sonication of lysate, the protein concentration was determined and aliquotted extracts (500 g/ml) were stored at –70C. Individual wells of a 96-well plate were coated with 50 ng EGFP or lenti packaging protein in carbonate buffer, pH 9.6, overnight at 4C. After washing three times in PBS-T, wells were incubated with blocking solution (BSA/PBS) for 2 h at 25C. Plasma samples were serially diluted in BSA/PBS-T. Wells were washed, and diluted plasma loaded in duplicate using 100 l/well. Wells were incubated with plasma for 2 h at 25C, washed three times, and incubated in 100 l goat anti-monkey IgG peroxidase-conjugated antibody (diluted in BSA/PBS-T; KPL, Gaithersburg, MD) for 1 h at 25C. The tetramethylbenzidine liquid substrate system (Sigma, St. Louis, MO) was used as substrate (30 min at 25C), and the reaction stopped with 0.5 M H2SO4 (50 l/well). Absorbency at 450 nm was recorded within 1 h. Data analysis. Means and standard errors of the mean (SEM) were calculated using Apple Macintosh systems with statistical software (Statview 512+, Brainpower Inc., Calabasas, CA). Data were compared with control values, where appropriate. Results were also compared with fetuses transferred with the HIV-1-derived lentiviral vector system IP at the same titer at 55 days gestation (n = 4), as reported [22].

ACKNOWLEDGMENTS We thank the animal technical and clinical laboratory staff at the CRPRC for their assistance. These studies were supported by NIH grants HL69748 and RR00169, and the UC Davis Children’s Miracle Network (A.F.T.). RECEIVED FOR PUBLICATION AUGUST 7; ACCEPTED OCTOBER 17, 2001.

REFERENCES 1. Fletcher, J. C., and Richter, G. (1996). Human fetal gene therapy. Hum. Gene Ther. 7: 1605–1614. 2. Senut, M.-C., and Gage, F. H. (1999). Prenatal gene therapy: can the technical hurdles be overcome? Mol. Med. Today 5: 152–156. 3. Zanjani, E. D., and Anderson, W. F. (1999). Prospects for in utero human gene therapy. Science 285: 2084–2088. 4. Boucher, R. C. (1999). Status of gene therapy for cystic fibrosis lung disease. J. Clin. Invest. 103: 441–445. 5. Davies, J. C., Geddes, D. M., and Alton, E. W. (1998). Prospects for gene therapy for cystic fibrosis. Mol. Med. Today 4: 292–299. 6. Ennist, D. L. (1999). Gene therapy for lung disease. Trends Pharmacol. Sci. 20: 260–266. 7. Goldman, M. J., Lee, P. S., Yang, J. S., and Wilson, J. M. (1997). Lentiviral vectors for gene therapy of cystic fibrosis. Hum. Gene Ther. 8: 2261–2268. 8. Jaffe, A., Bush, A., Geddes, D. M., and Alton, E. W. F. W. (1999). Prospects for gene therapy in cystic fibrosis. Arch. Dis. Child. 80: 286–289. 9. West, J., and Rodman, D. M. (2001). Gene therapy for pulmonary diseases. Chest 119: 613–617. 10. Jaffe, A., et al. (2000). Cationic lipid-mediated gene transfer to the growing murine and human airway. Gene Ther. 7: 273–278. 11. Khan, T. Z., et al. (1995). Early pulmonary inflammation in infants with cystic fibrosis. Am. J. Respir. Crit. Care Med. 151: 1075–1082. 12. Sturgess, J., and Imrie, I. (1982). Quantitative evaluation of the development of tracheal submucosal glands in infants with cystic fibrosis and control infants. Am. J. Pathol. 106: 303–311. 13. Ornay, A., et al. (1987). Pathological confirmation of cystic fibrosis in the fetus following prenatal diagnosis. Am. J. Med. Genet. 28: 935–947. 14. Kass Esler, A., et al. (1994). The impact of developmental stage, route of administration and the immune system on adenovirus-mediated gene transfer. Gene Ther. 1: 395–402. 15. Zepeda, M., and Wilson, J. M. (1996). Neonatal cotton rats do not exhibit destructive immune responses to adenoviral vectors. Gene Ther. 3: 973–979. 16. Robinson, C. B. (1998). Is DNA destiny? A cure for cystic fibrosis. Clin. Chest Med. 19: 527–534.

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17. Hislop, A., Howard, S., and Fairweather, D. V. I. (1984) Morphometric studies on the structural development of the lung in Macaca fascicularis during fetal and postnatal life. J. Anat. 138: 95–112. 18. Fanucchi, M. V., and Plopper, C. G. (1997). Pulmonary developmental responses to toxicants. In Comprehensive Toxicology. Vol. 8., Toxicology of the Respiratory System (I. G. Sipes, C. A. McQueen, and A. J. Gandolfi, Eds.), pp. 203–220. Elsevier Science Ltd., New York, NY. 19. Plopper, C. G., and Hyde, D. M. (1992). Epithelial cells of bronchioles. In Comparative Biology of the Normal Lung (R. A. Parent, Ed.), pp. 85–92. CRC Press, Boca Raton, FL. 20. Jeffrey, P., and Reid, L. (1977). Development of the Lung (W. A. Hodson, Ed.), pp. 87–134. Marcel Dekker, New York, NY. 21. Plopper, C. G., Alley, J. L., and Weir, A. J. (1986). Differentiation of tracheal epithelium during fetal maturation in the rhesus monkey Macaca mulatta. Am. J. Anat. 175: 59–71. 22. Tarantal, A. F., et al. (2001). Fetal rhesus monkey model for in utero gene transfer: studies with retroviral-based vector systems. Mol. Ther. 3: 128–138. 23. Tarantal, A. F., and Hendrickx, A. G. (1988). Prenatal growth in the cynomolgus and rhesus macaque (Macaca fascicularis and Macaca mulatta): a comparison by ultrasonography. Am. J. Primatol. 15: 309–323. 24. Li, J., et al. (2000). Assessment of humoral immune responses after retroviral vector gene transfer in vivo into fetal or neonatal rhesus monkeys. Blood 96: 805. 25. Cowan, M. J., Chou, S.-H., and Tarantal, A. F. (2001). Tolerance induction post in utero stem cell transplantation. In Stem Cells from Cord Blood, In Utero Stem Cell Development, and Transplantation-Inclusive Gene Therapy (W. Holzgreve and M. Lessl, Eds.), pp. 145–196. Springer-Verlag, New York, N.Y. 26. Kronquist, K. E., Crandall, B. F., and Cosico, L. G. (1984). Detection of novel fetal polypeptides in human amniotic fluid using two-dimensional gel electrophoresis. Tumour Biol. 5: 15–31. 27. Douar, A. M., et al. (1997). Foetal gene delivery in mice by intra-amniotic administration of retroviral producer cells and adenovirus. Gene Ther. 4: 883–890. 28. Douar, A. M., Themis, M., Sandig, V., Friedmann, T., and Coutelle, C. (1996). Effect of amniotic fluid on cationic lipid mediated transfection and retroviral infection. Gene Ther. 3: 789–796. 29. Holzinger, A., Trapnell, B. C., Weaver, T. E., Whitsett, J. A., and Iwamoto, H. S. (1995) Intraamniotic administration of an adenoviral vector for gene transfer to fetal sheep and mouse tissues. Pediatr. Res. 38: 844–850. 30. Senoo, M., et al. (2000). Adenovirus-mediated in utero gene transfer in mice and guinea pigs: tissue distribution of recombinant adenovirus determined by quantitative Taqmanpolymerase chain reaction assay. Mol. Gen. Metab. 69: 269–276. 31. Bestor, T. H. (2000). Gene silencing as a threat to the success of gene therapy. J. Clin. Invest. 105: 409–411. 32. Rivella, S., and Sadelain, M. (1998). Genetic treatment of severe hemoglobinopathies: the combat against transgene variegation and transgene silencing. Semin. Hematol. 35: 112–125. 33. Khoor, A., Stahlman, M. T., Gray, M. E., and Whitsett, J. A. (1994). Temporal-spatial distribution of SP-B and SP-C proteins and mRNAs in developing respiratory epithelium of human lung. J. Histochem. Cytochem. 42: 1187–1199. 34. Wert, S. E., Glasser, S. W., Korfhagen, T. R., and Whitsett, J. A. (1993). Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice. Dev. Biol. 156: 426–443. 35. Whitsett, J. A., et al. (1992). Human cystic fibrosis transmembrane conductance regulator directed to respiratory epithelial cells of transgenic mice. Nat. Genet. 2: 13–20. 36. Tarantal, A. F., and Gargosky, S. E. (1995). Characterization of the insulin-like growth factor (IGF) axis in the serum of maternal and fetal macaques (Macaca mulatta and Macaca fascicularis). Growth Reg. 5: 190–198. 37. Naldini, L., et al. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272: 263–267. 38. Zufferey, R., Nagy, D., Mandel, R.J., Naldini, L., and Trono, D. (1997). Multiple attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol. 15: 871–875. 39. Tarantal, A. F. (1990). Interventional ultrasound in pregnant macaques: embryonic/fetal applications. J. Med. Primatol. 19: 47–58. 40. Tarantal, A. F. (1993). Hematologic reference values for the fetal long-tailed macaque (Macaca fascicularis). Am. J. Primatol. 29: 209–219. 41. Tarantal, A. F., et al. (1995). Effects of viral virulence on intrauterine growth in SIVinfected fetal rhesus macaques (Macaca mulatta). J. AIDS Hum. Retrovirol. 10: 129–138. 42. Tarantal, A. F., Goldstein, O., Barley, F., and Cowan, M. J. (2000) Transplantation of human peripheral blood stem cells (PBSC) into fetal rhesus monkeys (Macaca mulatta). Transplantation 69: 1818–1823. 43. Lockridge, K. M., et al. (1999). Pathogenesis of experimental rhesus cytomegalovirus infection. J. Virol. 73: 9576–9583.

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