Fetal Gene Transfer Research Articles

Feasibility of Retroviral Vector-Mediated in utero Gene Transfer to the Fetal Rabbit

Objectives: Successful treatment or prevention of severe hereditary diseases could conceivably be achieved by genetic intervention early in development. Viral vector-mediated fetal gene transfer is proving a valuable tool to test the above concept in relevant animal models. Although the pregnant rabbit is a well-recognized model for fetal therapy, few preclinical assays have used it to validate fetal gene transfer approaches. In this preliminary study we assessed for the first time the feasibility of retroviral vector-mediated in utero gene transfer in the fetal rabbit. Methods: Different amounts of the vesicular stomatitis virus G pseudotyped MFG(nls)LacZ retroviral vector, expressing a nuclear-localized β-galactosidase reporter protein were injected intraperitoneally and -hepatically into 20- to 22-day-old fetuses. At 8–9 days post-treatment, the pups were sacrificed and the tissues harvested for analysis. Evidence of gene transfer was obtained by PCR amplification of proviral sequences within genomic DNA isolated from the treated samples. Transgenic β-galactosidase expression was assessed by X-gal histochemical staining. Results: By intraperitoneal injection 43% of the viable fetuses treated (3/7) showed evidence of successful LacZ gene transfer and low-level β-galactosidase expression into liver and heart, whereas by intrahepatic injection roughly 38% (3/8) of the livers were positive for LacZ gene transfer and expression. The success rate for the viable fetuses rose to 67% positive livers (4/6) when a near double amount of recombinant virus was injected using a 10-fold concentrated virus stock. In terms of short-term safety, fetal and maternal survival rates approached 80% of treated fetuses, and 100% of treated does. Conclusions: The pregnant rabbit is a useful and reliable model allowing the design of further studies to optimize the conditions for effective, safer, and persistent retroviral vector-mediated fetal gene transfer.

Intrapulmonary and intramyocardial gene transfer in rhesus monkeys (Macaca mulatta): Safety and efficiency of HIV-1-derived lentiviral vectors for fetal gene delivery

Fetal gene transfer was studied using intrapulmonary and intramyocardial transfer of SIN HIV-1-derived lentiviral vectors expressing EGFP in rhesus monkeys. Fetuses were monitored sonographically during gestation and tissue analyses performed at term or 3 months postnatal age. Animals remained healthy during the study period as evidenced by normal growth, development, hematology, clinical chemistry, echocardiography, and pulmonary function tests. Strong pulmonary fluorescence was observed postnatally after fetal intrapulmonary delivery of lenti-CMV, but not lenti-SP-C, and compared to nontransferred controls. High EGFP copy numbers were found by quantitative PCR with both vector constructs in lung lobes (<or=15%) and EGFP copies were also detected in the diaphragm, pericardium, and thorax. No differences were found in lung:body weight ratios, percentage lung parenchyma, or overall morphology when compared to controls. For intramyocardial gene delivery, strong transgene expression was found within the myocardium and pericardium, and high EGFP copy numbers were found by quantitative PCR (3-36%). EGFP was also detected in the aorta, thorax, and diaphragm. These studies indicate that postnatal heart and lung development and function were not altered after fetal intraorgan gene transfer and subsequent transgene expression prenatally and postnatally, and gene transfer was restricted to the thoracic cavity with intrapulmonary and intramyocardial lentiviral vector-mediated gene delivery.

125. AAV Vector-Mediated Neonatal Gene Transfer: Efficient Transgene Expression in Muscles after Intraperitoneal Cavity Vector Injection

Neonatal or fetal gene transfer is a useful approach to treat genetic diseases which cause irreversible manifestations in adulthood, and is also advantageous in transducing tissues at relatively low vector doses. Among the routes of administration, intraperitoneal cavity injection is potentially useful for neonates as it is possible to administer larger amount of vector stocks under easier and safer circumstances. In this study, we explored the optimal use of AAV vectors administered into intraperitoneal cavity for neonatal gene therapy in mice. For this purpose, vectors carrying human coagulation factor IX (hFIX) gene under cytomegalovirus (CMV)-derived promoter using AAV1-5 capsids were injected intraperitoneally into immune-competent (C57BL/6) mice within 24 hours after birth. Each neonate mouse received a dose of 1|[times]|1010 genome copy per body and plasma concentrations of hFIX were monitored. As a result, AAV1 vector injection group showed the highest hFIX concentration (195.3|[plusmn]|60.2 ng/ml), followed by AAV5 (11.8|[plusmn]|1.8 ng/ml) and AAV2 (3.7|[plusmn]|6.5 ng/ml) groups. The concentration of hFIX observed after intraperitoneal AAV1 injection was slightly higher than the group with intramuscular injection of AAV1 at the same dose. Dose escalation is feasible for intraperitoneal cavity injection up to 1|[times]|1011 genome copy per body and the result showed a marked increase in hFIX concentration (613.5|[plusmn]|223.5 ng/ml), a level that is considered within fully therapeutic range. The hFIX concentration remained constant for more than 24 weeks. To analyze tissue distribution of expression, AAV1 vectors encoding LacZ or luciferase were administered intraperitoneally and analyzed by X-Gal staining or bioluminescence imaging analysis using IVIS system, respectively. Interestingly, both results showed transgene expression in muscles of trunk and proximal limbs adjacent to the peritoneal tissue in addition to peritoneal epithelium. Analysis of organ specific expression after sacrifice confirmed expression in muscles of thorax, thigh, brachial and iliopsoas region. Muscle mediated expression appear to contribute to high level of transgene expression even the vectors were administered intraperitoneally. These findings support the efficacy of neonatal gene therapy and would guide to design strategies for neonatal gene therapy using AAV vectors.

HIV-1-derived lentiviral vectors and fetal route of administration on transgene biodistribution and expression in rhesus monkeys

The gene transfer efficiency of lentiviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein (VSV-G) driven by the MND or CMV promoters and expressing the enhanced green fluorescent protein (EGFP) was investigated in fetal rhesus monkeys (Macaca mulatta) (N=21). Fetuses (50+/-10 days gestation; term 165+/-10 days) were injected under ultrasound guidance using an intraperitoneal (i.p.) or intrahepatic (i.h.) approach with a range of 1 x 10(7)-2 x 10(8) infectious particles/fetus. Analysis of transgene biodistribution and expression was performed in multiple tissues at 3-7 months postgene delivery using quantitative techniques. Overall, results indicated the following: (1) i.p. gene transfer at 40 days gestation resulted in a more diffuse distribution of the vector compared to administration at 60 days gestation; (2) vector biodistribution was similar after administration by the i.p. or i.h. routes; and (3) gene expression analysis in transduced tissues showed the presence of mRNA transcripts that correlated with the level of gene transfer. These studies suggest that fetal gene transfer using the i.p. and i.h. routes results in prolonged transduction and expression of the transgene in multiple tissues.

987. Highly Efficient Regulation of Gene Expression by Tetracycline in a Novel Replication-Defective Herpes Simplex Viral Vector

Fetal gene transfer is a promising therapy for the correction of pathology associated with inherited or acquired diseases, however information on the optimal conditions for gene transfer is limited. In this study, we have determined the effect of the time of gene transfer and route of administration on the efficiency of this approach. Fetal rhesus monkeys were injected intraperitoneal (IP) at 40 (N=4) or 60 (N=4) days of gestation (term 165±10 days) with 2 × 107 HIV-1-derived lentiviral vector particles/fetus (HIV/VSV-G/MND/EGFP) and gene marking and expression was assessed in multiple tissues (cerebrum, trachea, esophagus, thymus, lung, heart, pericardium, thorax, lymph nodes, liver, spleen, pancreas, kidneys, adrenals, gonads, stomach, colon, diaphragm, omentum, peritoneum, bone marrow) using quantitative real-time PCR. Results were compared to lentiviral vector gene transfer using the intrahepatic (IH) (N=4) and intraportal (Ipo) (N=4) routes of administration. Analysis revealed that gene transfer at 40 days gestation resulted in high gene marking in omentum (18%), peritoneum (1%) and diaphragm (5%) with levels up to 400-fold higher than in other abdominal organs. The amount of gene expression highly correlated (P < 0.0001) with the extent of gene transduction within the peritoneal cavity. We obtained similar vector biodistribution at 60 days gestation, and no significant differences in the amount of gene transfer (P < 0.005) were found between the groups. However, gene transfer and expression at 40 versus 60 days gestation in nonabdominal tissues were 3 - 20 and 4 - 400 - fold higher, respectively. When comparing IH to IP gene delivery, a similar vector biodistribution was found with no significant differences on the extent of gene transfer (P < 0.05) in all tissues examined. In contrast, Ipo administration resulted in a reduction of vector spread and a 200-fold reduction in the level of gene marking when compared to the IP route of administration. In conclusion, definition of the conditions at the time of fetal transfer will further shape the feasibility and safety of this approach for the treatment of human disease.

Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle.

271. Fetal Gene Transfer Using Lentiviral Vectors for Heart, Lung, and Blood Diseases: Assessments of Safety and Efficiency in Rhesus Monkeys

271. Fetal Gene Transfer Using Lentiviral Vectors for Heart, Lung, and Blood Diseases: Assessments of Safety and Efficiency in Rhesus Monkeys

Fetal gene transfer by intrauterine injection with microbubble-enhanced ultrasound.

Intrauterine injection of naked DNA expressing luciferase, green fluorescent protein (GFP), or beta-galactosidase (beta-gal) and fluorescein isothiocyanate-labeled oligodeoxynucleotide (FITC-ODN), in combination with microbubble-enhanced ultrasound (US), referred to as the "shotgun method" (SGM), produced high-level protein expression in fetal mice. With the SGM, luciferase expression increased approximately 10(3)-fold in comparison with expression after injection of naked DNA alone. Electron microscopic analysis demonstrated transient formation of pores on the skin surface after intraamniotic (i.a.) injection with the SGM. Widespread expression of GFP and beta-gal and delivery of FITC-ODN were observed in multiple fetal tissues adjacent to the injection points. PCR analysis indicated that germline transfection was only transient following intraperitoneal (i.p) injection, and there was no evidence of transfer of the reporter gene to the offspring. Thus, SGM might provide a useful means to clarify the molecular mechanisms of genetic diseases in utero, as well as a tool to develop gene therapies in utero.

Systemic Diffusion Including Germ Cells after Plasmidic in utero Gene Transfer in the Rat

Objectives: The integration of a proviral plasmid into the host genome could be a good approach for fetal somatic gene transfer. The goal of this study was to assess the integration and transcription of a proviral marker gene injected intraperitoneally into rat fetuses as well as the risks of maternal contamination and germ-line transmission. Methods: On day 17 post-coitus, each fetus was injected intraperitoneally with 10 µg plasmid DNA through the uterine wall. Twenty-one days after spontaneous delivery, integration and transcription of the plasmid in gonad, gut, liver, spleen, lung and brain tissue from 10 pups were determined by PCR and RT-PCR. Results: 14 of 60 organs exhibited integration of the plasmid. Four samples of gut (40%), 3 samples of liver and spleen (30%), 2 samples of brain (20%) and no sample of lung were transfected. Two testicular samples were transfected and study of F1 rats from 2 brothers of one of the positive rats revealed transgenic pups from 1 of these 2 animals. No transfection of maternal tissues was detected. Conclusion: Integration and transcription of a marker gene injected intraperitoneally into rat fetuses appear efficient, especially in intraperitoneal organs. The risk of maternal contamination appears very low when using a naked DNA plasmid injected directly into fetuses. However, germ-line contamination can occasionally occur even with injection late during pregnancy, suggesting further studies are necessary to assess this risk in direct gene transfer experiments.

Lentiviral vector gene transfer into fetal rhesus monkeys (Macaca mulatta): lung-targeting approaches.

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 x 10(7) 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 (< or = 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.

Fetal gene transfer by transuterine injection of cationic liposome-DNA complexes.

In utero injection of cationic liposome-DNA complexes (CLDCs) containing chloramphenicol acetyltransferase, beta-galactosidase (beta-gal), or human granulocyte colony-stimulating factor (hG-CSF) expression plasmids produced high-level gene expression in fetal rats. Tissues adjacent to the injection site exhibited the highest levels of gene expression. Chloramphenicol acetyltransferase expression persisted for at least 14 days and was reexpressed following postnatal reinjection of CLDCs. Intraperitoneal administration of the hG-CSF gene produced high serum hG-CSF levels. X-gal staining demonstrated widespread beta-gal expression in multiple fetal tissues and cell types. No toxic or inflammatory responses were observed, nor was there evidence of fetal-maternal or maternal-fetal gene transfer, suggesting that CLDCs may provide a useful alternative to viral vectors for in utero gene transfer.

A new vector for fetal gene transfer

A new vector for fetal gene transfer

Prenatal gene therapy: can the technical hurdles be overcome?

Fifty years ago, a medical breakthrough in the prenatal diagnosis of genetic disorders was made with the introduction of amniocentesis. Until recently, there was little hope that diseases diagnosed in utero could be treated before birth. Today, prenatal gene therapy is emerging as a new concept for treating pre- and postnatal manifestations of genetic diseases and developmental disorders. Research studies have generated a degree of optimism by demonstrating the feasibility of fetal gene transfer. Nevertheless, enthusiasm is tempered by the considerable technical and ethical issues raised by such studies. Undoubtedly, the future of prenatal gene transfer as a therapeutic approach for birth defects mostly depends on addressing and overcoming these concerns.

Long-Term Transgene Expression in Fetal Rat Suprachiasmatic Nucleus Neurografts Followingex VivoAdenoviral Vector-Mediated Gene Transfer

Ex vivogene transfer to fetal suprachiasmatic nucleus (SCN)-containing solid piece neurografts was explored using a first-generation prototype adenoviral vector containing the reporter gene LacZ (Ad–LacZ). Transgene expression was examined at different intervals following grafting in the IIIrd ventricle of rat brain and was compared to that of explant cultures. Large numbers of β-galactosidase-positive cells were observed 8 days postgrafting. The number of stained cells had decreased considerably at 21 days but transduced cells were still present at 70 days.In vitroculturing of infected SCN tissue revealed high expression up to 21 days, indicating that thein vivoandin vitrofates of Ad–LacZ-infected cells were different. The main reason for this difference appeared to be cell loss by necrosis in the initial phase after transplantation, a phenomenon not related to the infection with Ad–LacZ since it similarly occurred in control grafts.In vivoinflammatory responses, observed after immunostaining for macrophages and T-lymphocytes, were also comparable in control and Ad-LacZ-treated transplants, except that cytotoxic T-cells were observed in the Ad–LacZ-treated transplants and not in controls. The recruitment of these cells was, however, minor and primarily observed at 8 days postgrafting, indicating that a major immunological rejection of the transduced graft did not occur. In both control and Ad–LacZ-infected transplants similar survival and intraimplant neuritic growth of SCN cells were visible.Ex vivogene transfer of solid piece fetal SCN grafts with adenoviral vectors therefore appeared to be a nontoxic long-term gene-introducing procedure. This would in principle enable the local production of neurotrophic factors within the transplant and has the potential to improve functional SCN neurografting.

Fetal somatic gene therapy.

Fetal somatic gene therapy is emerging as a new experimental approach, in particular to prevent irreversible perinatal disease manifestation for many inherited conditions. Early therapeutic gene application may also allow targeting of still expanding stem cell populations of organ or cell systems inaccessible later in life and help to avoid immune sensitization against the therapeutic vector system or transgene protein product. The progress in development of ultrasound scanning and embryofetoscopy over the last decade has made minimally invasive administration of therapeutic gene transfer vectors to the fetus in utero possible in principle. We review here the different considerations in choosing candidate diseases, the possible routes of administration and times in fetal development for application of a therapeutic gene and discuss the benefits and problems of present vector systems in this context. Given the many unknown aspects of fetal gene transfer, it is essential to extensively investigate this new approach to gene therapy in animal models for specific diseases, to improve on the technology of delivery and to assess efficacy of expression as well as the possible side effects before application to humans can be considered.