Fetal echocardiographic assessment of cardiovascular impact of prolonged support on EXTrauterine Environment for Neonatal Development (EXTEND) system.

OBJECTIVE: EXTrauterine Environment for Neonatal Development (EXTEND) is a system to support ongoing fetal growth and organ development in an extrauterine environment, utilizing a pumpless low-resistance oxygenator circuit. The aim of this study was to evaluate hemodynamics and cardiac function in fetal sheep sustained on the EXTEND system. METHODS: This was a prospective study of fetal sheep supported for a minimum of 3 weeks on EXTEND. Hemodynamic parameters were assessed weekly and included heart rate, mean arterial pressure (MAP), Doppler-echocardiography-derived cardiac output (CO), pulsatility indices (PIs) of the fetal middle cerebral artery (MCA), umbilical artery (UA) and ductus venosus and cardiac function, as assessed by speckle-tracking-derived global longitudinal strain and strain rate in the right (RV) and left (LV) ventricles. Parameters were compared at 0 days and 1, 2 and 3 weeks following placement on EXTEND. RESULTS: Of 10 fetal sheep enrolled, seven survived for 3 weeks and were included in the analysis. Median gestational age at cannulation was 107 (range, 95-109) days. Heart rate decreased and MAP increased significantly, but within acceptable ranges, during the study period. The quantities and relative ratios of right and left CO remained stable within the anticipated physiological range throughout the study period. Vascular tracings and PIs appeared to be similar to those seen normally in the natural in-utero state, with MCA-PI being higher than UA-PI. UA tracings demonstrated maintained abundant diastolic flow despite the absence of placental circulation. In both the RV and LV, strain decreased significantly at 1 and 2 weeks relative to baseline but returned to baseline values by week 3. CONCLUSIONS: The EXTEND mechanical support system replicates natural physiology and creates a stable and sustainable cardiovascular construct that supports growth over a 3-week period. However, there is a period of depressed contractility within the first week with subsequent improvement by week 3. This may reflect a period of physiological accommodation that warrants further investigation. This study lays the foundation for further exploration as the EXTEND system moves towards human application. © 2019 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

Physician views regarding the benefits and burdens of prenatal surgery for myelomeningocele.

OBJECTIVE: Examine how pediatric and obstetrical subspecialists view benefits and burdens of prenatal myelomeningocele (MMC) closure. STUDY DESIGN: Mail survey of 1200 neonatologists, pediatric surgeons and maternal-fetal medicine specialists (MFMs). RESULTS: Of 1176 eligible physicians, 670 (57%) responded. Most respondents disagreed (68%, 11% strongly) that open fetal surgery places an unacceptable burden on women and their families. Most agreed (65%, 10% strongly) that denying the benefits of open maternal-fetal surgery is unfair to the future child. Most (94%) would recommend prenatal fetoscopic over open or postnatal MMC closure for a hypothetical fetoscopic technique that had similar shunt rates (40%) but decreased maternal morbidity. When the hypothetical shunt rate for fetoscopy was increased to 60%, physicians were split (49% fetoscopy versus 45% open). Views about burdens and fairness correlated with the likelihood of recommending postnatal or fetoscopic over open closure. CONCLUSION: Individual and specialty-specific values may influence recommendations about prenatal surgery.

Longitudinal neurodevelopmental and neuromotor outcome in congenital diaphragmatic hernia patients in the first 3 years of life.

OBJECTIVE: The objective of this study was to longitudinally evaluate the neurodevelopmental (ND) outcome in congenital diaphragmatic hernia (CDH) survivors during the first 3 years of life. STUDY DESIGN: The study cohort consists of 47 CDH survivors that were enrolled in our prospective, follow-up program between July 2004 and September 2010, and underwent serial ND evaluations during the first 3 years of life. ND outcomes were evaluated using the Bayley Scales of Infant Development (BSID)-II or BSID-III. Persistent ND impairment was defined as a score that remained 79 for the cognitive, language and psychomotor domains at the most recent follow-up visit compared with the first assessment. RESULT: The median age at first and last evaluation was 8 (range, 5 to 15) and 29 (range, 23 to 36) months, respectively. During the follow-up, ND scores improved to average in 17%, remained average in 60%, remained delayed in 10%, improved from severely delayed to mildly delayed in 2% and deteriorated from average to delayed in 15%. Motor scores improved to average in 26%, remained average in 55%, remained delayed in 8% and improved from severely delayed to mildly delayed in 11%. Intrathoracic liver position (P=0.004), preterm delivery (P=0.03), supplemental O2 requirement at day of life 30 (P=0.007), age at discharge (P=0.03), periventricular leukomalacia (PVL; P=0.004) and initial neuromuscular hypotonicity (P=0.01) were associated with persistent motor delays. No relationship was found between patient's characteristics and the risk of persistent cognitive and language delays. CONCLUSION: (1) The majority of children with CDH are functioning in the average range by early preschool age, (2) most children who had early delays showed improvement in their ND outcome, (3) children showing delays in all the three domains were the least likely to show improvement and (4) CDH severity appears to be predictive of persistent psychomotor delays.

Early gestational gene transfer with targeted ATP7B expression in the liver improves phenotype in a murine model of Wilson's disease.

The ideal gene therapy for metabolical liver disorders would target hepatocytes before the onset of disease and be durable, non-toxic and non-immunogenic. Early gestational gene transfer can achieve such goals. Here, we demonstrate that prenatal gene transfer of human Atp7b reduces liver pathology and improves biochemical markers in Atp7b(-/-) mice, a murine model of Wilson's disease (WD). Following prenatal injection of lentivirus vector containing the human Atp7b gene under the transcriptional control of a liver-specific promoter, the full-length ATP7B was detectable in mouse livers for the entire duration of experiments (20 weeks after birth). In contrast to a marked pathology in non-injected animals, livers from age-matched treated mice consistently demonstrated normal gross and histological morphology. Hepatic copper content was decreased in the majority of treated mice, although remaining copper levels varied. Improvement of hepatic copper metabolism was further apparent from the presence of copper-bound ceruloplasmin in the sera and normalization of the mRNA levels for HMG CoA-reductase. With this approach, the complete loss of copper transport function can be ameliorated, as evident from phenotypical improvement in treated Atp7b(-/-) mice. This study provides proof of principle for in utero gene therapy in WD and other liver-based enzyme deficiencies.

Early intra-amniotic gene transfer using lentiviral vector improves skin blistering phenotype in a murine model of Herlitz junctional epidermolysis bullosa.

Mutations of the LAMB3 gene cause a lethal form of junctional epidermolysis bullosa (JEB). We hypothesized that early intra-amniotic gene transfer in a severe murine model of JEB would improve or correct the skin phenotype. Time-dated fetuses from heterozygous LAMB3(IAP) breeding pairs underwent ultrasound guided intra-amniotic injection of lentiviral vector encoding the murine LAMB3 gene at embryonic day 8 (E8). Gene expression was monitored by immunohistochemistry. The transgenic laminin-ß3 chain was shown to assemble with its endogenous partner chains, resulting in detectable amounts of laminin-332 in the basement membrane zone of skin and mucosa. Ultrastructually, the restoration of ∼60% of hemidesmosomal structures was also noted. Although we could correct the skin phenotype in 11.9% of homozygous LAMB3(IAP) mice, none survived beyond 48 h. However, skin transplants from treated E18 homozygous LAMB3(IAP) fetuses maintained normal appearance for 6 months with persistence of normal assembly of laminin-332. These results demonstrate for the first time long-term phenotypic correction of the skin pathology in a severe model of JEB by in vivo prenatal gene transfer. Although survival remained limited due to the limitations of this mouse model, this study supports the potential for treatment of JEB by prenatal gene transfer.

Jaagsiekte sheep retrovirus pseudotyped lentiviral vector-mediated gene transfer to fetal ovine lung.

Viral vector-mediated gene transfer to the postnatal respiratory epithelium has, in general, been of low efficiency due to physical and immunological barriers, non-apical location of cellular receptors critical for viral uptake and limited transduction of resident stem/progenitor cells. These obstacles may be overcome using a prenatal strategy. In this study, HIV-1-based lentiviral vectors (LVs) pseudotyped with the envelope glycoproteins of Jaagsiekte sheep retrovirus (JSRV-LV), baculovirus GP64 (GP64-LV), Ebola Zaire-LV or vesicular stomatitis virus (VSVg-LV) and the adeno-associated virus-2/6.2 (AAV2/6.2) were compared for in utero transfer of a green fluorescent protein (GFP) reporter gene to ovine lung epithelium between days 65 and 78 of gestation. GFP expression was examined on day 85 or 136 of gestation (term is ∼145 days). The percentage of the respiratory epithelial cells expressing GFP in fetal sheep that received the JSRV-LV (3.18 × 10(8)-6.85 × 10(9) viral particles per fetus) was 24.6±0.9% at 3 weeks postinjection (day 85) and 29.9±4.8% at 10 weeks postinjection (day 136). Expression was limited to the surface epithelium lining fetal airways <100 µm internal diameter. Fetal airways were amenable to VSVg-LV transduction, although the percentage of epithelial expression was low (6.6±0.6%) at 1 week postinjection. GP64-LV, Ebola Zaire-LV and AAV2/6.2 failed to transduce the fetal ovine lung under these conditions. These data demonstrate that prenatal lung gene transfer with LV engineered to target apical surface receptors can provide sustained and high levels of transgene expression and support the therapeutic potential of prenatal gene transfer for the treatment of congenital lung diseases.

Early gestational gene transfer of IL-10 by systemic administration of lentiviral vector can prevent arthritis in a murine model.

Gene therapy has been applied to murine models of rheumatoid arthritis (RA) using a number of different strategies to downregulate inflammation in synovial joints. However, prolonged joint expression has been problematic. Our laboratory has found that early gestational intravascular injection of lentiviral vector leads to efficient transduction and sustained transgene expression in articular cartilage and synovium. In this study, we show that in utero gene transfer of IL-10 can prevent and decrease pathology in a murine model of RA. Following prenatal injection of lentiviral vector containing murine IL-10 gene, the cytokine was detectable in the serum, and the green fluorescent protein reporter gene was detectable in chondrocytes and synoviocytes of adult mice up to 21 weeks of age. Adult mice that had been treated prenatally were later immunized against type II collagen to induce an autoimmune arthritis. Compared with controls, prenatally treated mice demonstrated delayed onset of arthritis, decreased frequency of arthritis and markedly decreased severity of disease, by both clinical and histological criteria. This effect was directly related to levels of IL-10 expression, but no immunosuppressive effects of the therapy were observed. This study demonstrates proof of principle for the prenatal prevention and amelioration of RA by early gestational gene transfer of the anti-inflammatory cytokine, IL-10.

Induction of fetal diuresis with intraamniotic furosemide injection reduces intestinal damage in a rat model of gastroschisis.

BACKGROUND/PURPOSE: Contact with amniotic fluid causes intestinal damage (ID) in fetuses with gastroschisis. Intraamniotic meconium has been shown to be responsible for ID, and ID has been shown to correlate with intraamniotic meconium concentrations. ID can be prevented by lowering the intraamniotic meconium concentration. A new method to lower intraamniotic meconium concentration might consist in the induction of fetal diuresis with intraamniotic diuretic injection. This hypothesis was tested in a rat model. MATERIALS AND METHOD: There were 4 experimental groups. CONTROL GROUP: Rat fetuses without any manipulation. Fetuses were harvested by cesarean section for examination at E21.5 (Term). SHAM GROUP: On E18.5, the hind limb of the rat fetuses were exteriorized by hysterotomy and replaced in the uterus. GASTROSCHISIS GROUP: Gastroschisis was surgically created in rat fetuses on E18.5, under a dissection microscope (16×). GASTROSCHISIS+FUROSEMIDE GROUP: After surgical creation of gastroschisis on E18.5, intraamniotic furosemide (5 mg/kg) was administered to the fetuses on E20. All fetuses were harvested on E21.5. RESULTS: There was no significant difference between intestinal serosal thicknesses of the control and sham groups. The serosal thickness was significantly higher in the gastroschisis group compared to the control group. In the gastroschisis+furosemide group, the intestinal serosal thickness was found significantly decreased compared with the gastroschisis group. CONCLUSION: Intraamniotic furosemide injection caused a substantial decrease in ID encountered in gastroschisis. The induction of fetal diuresis with intraamniotic furosemide injection seems promising as a prenatal treatment modality.

SLAM-enriched hematopoietic stem cells maintain long-term repopulating capacity after lentiviral transduction using an abbreviated protocol.

Gene transfer to long-term repopulating hematopoietic stem cells (HSCs) using integrating viral vectors is an important goal in gene therapy. The SLAM (signaling lymphocyte activation molecule)-family receptors have recently been used for the isolation of highly enriched murine HSCs. This HSC enrichment protocol is relatively simple, and results in an HSC population with comparable repopulating capacity to c-kit(+)lin(-)Sca-1(+) (KSL) HSCs. The capacity to withstand genetic manipulation and, most importantly, to maintain long-term repopulating capacity of SLAM-enriched HSC populations has not been reported. In this study, SLAM-enriched HSCs were assessed for transduction efficiency and in vivo long-term repopulating capacity after lentiviral transduction using an abbreviated transduction protocol and KSL-enriched HSCs as a reference population. SLAM- and KSL-enriched HSCs were efficiently transduced by lentiviral vector using a simple protocol that involves minimal in vitro manipulation and no pre-stimulation. SLAM-HSCs are at least equal to KSL-HSCs with respect to efficiency of transduction and maintenance of long-term repopulating capacity. Although there was a reduction in repopulating capacity related to enrichment and culture manipulations relative to freshly isolated bone marrow (BM) cells, no detrimental effects were identified on long-term competitive capacity related to transduction, as transduced cells maintained stable levels of chimerism in competition with non-transduced cells and freshly isolated BM cells. These results support the SLAM-HSC enrichment protocol as a simple and efficient method for HSC enrichment for gene transfer studies.

The developmental stage determines the distribution and duration of gene expression after early intra-amniotic gene transfer using lentiviral vectors.

Gene transfer after intra-amniotic injection has, in general, been of low efficiency and limited to epithelial cells in the skin, pulmonary and gastrointestinal system. We have recently shown that early gestational administration results in a more efficient gene transfer to developmentally accessible stem cell populations in the skin and eye. In this study we present a comprehensive analysis of patterns of tissue expression seen after early intra-amniotic gene transfer (IAGT) using lentiviral vectors. To assess the influence of developmental stage on tissue expression, injections were administered from the late head fold/early somite stage (E8) to E18. In early gestation (E8-10), green fluorescent protein (GFP) expression was observed in multiple organs, derived from all three germ layers. Remarkably, GFP expression was observed in tissues derived from mesoderm and neural ectoderm at E8, whereas expression was limited to only epithelial cells of ectoderm- and endoderm-derived organs after E11. The amount and duration of gene expression was much higher after IAGT at early gestational time points. The observed temporal patterns of gene expression correspond to the predicted developmental accessibility of organ-specific cell populations. This model may be useful for the analyses of mechanisms of genetic and/or developmental disease and for the development of prenatal gene therapy for specific disorders.

Retinoic acid induced myelomeningocele in fetal rats: characterization by histopathological analysis and magnetic resonance imaging.

The prevention of human neural tube defects by folic acid administration and the potential for fetal surgical intervention for myelomeningocele (MMC) have renewed interest in the molecular pathways and pathophysiology of spina bifida. Animal models for assessment of the early developmental biology and pathophysiology of this lesion are needed. The goal of this study was to develop and characterize a non-surgical rat model of MMC. Time-dated Sprague-Dawley rats were gavage fed different doses of retinoic acid (RA) dissolved in olive oil at E10 (maternal n = 55, fetal n = 505). Control animals received olive oil alone (maternal n = 20, fetal n = 265) or were untreated (maternal n = 5, fetal n = 63). Fetuses were analyzed by detailed histopathology and MRI. Overall, isolated MMC occurred in 60.7% (307/505) of RA-exposed fetuses and no controls. Histopathology confirmed the entire spectrum of severity observed in human MMC, ranging from exposure of the cord with intact neural elements to complete cord destruction. MRI of the brain of MMC fetuses confirmed structural changes similar to humans with Arnold-Chiari malformation, including downward displacement of the cerebellum to just above the foramen magnum and compression of the developing medulla into a small posterior fossa. In conclusion, the RA-induced rat model of MMC is developmentally and anatomically analogous to human MMC. This relatively efficient and cost-effective model of MMC should facilitate investigation of the developmental biology and pathophysiology of MMC, and may be useful for the evaluation of further strategies for prenatal treatment.

Fetal head biometry following in-utero repair of myelomeningocele.

OBJECTIVE: To evaluate the impact of prenatal myelomeningocele repair on fetal head biometry. METHODS: Fifty fetuses underwent open fetal myelomeningocele repair at our institution between January 1998 and July 2002. All had serial head circumference (HC) and lateral ventricular diameter (VD) measurements taken preoperatively and weekly for 8 weeks after repair. Cortical index (CI) was defined as HC/VD. Measurements were compared with gestational age-matched values from nomograms. One-sample t-test, ANOVA and repeated measures analysis were used to assess HC, VD and CI after fetal repair. RESULTS: Preoperatively, the HC in fetuses with myelomeningocele was smaller than control values (186.4 vs. 198.8 mm, P = 0.0004). Eight weeks' postoperatively this difference had resolved (293 vs. 301.6 mm, P = 0.76). The mean increase in CI after repair was 20% (P = 0.02) compared with the predicted 51% in normal cases. The average increase in VD was 3.9 mm (38.8%, P < 0.001). CONCLUSIONS: Mid-gestational repair of myelomeningocele alters fetal head growth. Increased CI suggests HC changes are not due to ventriculomegaly alone.

Gastroschisis in the rat model is associated with a delayed maturation of intestinal pacemaker cells and smooth muscle cells.

BACKGROUND: A pacemaker system is required for peristalsis generation. The interstitial cells of Cajal (ICC) are considered the intestinal pacemaker, and are identified by expression of the c-kit gene--encoded protein. Gastroschisis is characterized by a severe gastrointestinal dysmotility in newborns. In spite of this clinical picture, few studies have focused on smooth muscle cells (SMC) morphology and none on ICC. Therefore, their morphology has been studied in fetuses at term in the rat model of gastroschisis. METHODS: At 18.5 day's gestation (E18.5), 10 rat fetuses were killed, 10 underwent surgical creation of gastroschisis, and 10 underwent manipulation only. The small intestine of the latter 2 groups was harvested at E21.5. Specimens were processed for H&E, c-kit and actin (alpha smooth muscle antibody [alpha-SMA]) immunohistochemistry, and transmission electron microscopy (TEM). RESULTS: In the controls, SMC were c-kit+ and alpha-SMA+, with labeling intensity increasing by age. At E21.5, some cells around the Auerbach's plexus were more intensely c-kit+, and differentiating ICC were seen under TEM at this level. Gastroschisis fetuses had no c-kit+ cells referable to ICC. In the more damaged loops, SMC were very faintly c-kit+ and alpha-SMA+. Under TEM, there were few differentiated SMC and no presumptive ICC. In the less-damaged loops, SMC were faintly c-kit+ and alpha-SMA+ and had ultrastructural features intermediate between those of E18.5 and E21.5 controls; ICC were very immature. CONCLUSIONS: ICC and SMC differentiation is delayed in gastroschisis with the most damaged loops showing the most incomplete picture. These findings might help in understanding the delayed onset of peristalsis and the variable time-course of the recover seen in babies affected by gastroschisis.

Neuronal differentiation and myenteric plexus organization are delayed in gastroschisis: an immunohistochemical study in a rat model.

Gastroschisis is a malformation due to prenatal rupture of the abdominal wall and evisceration of the midgut. Intestinal loops are shortened, matted, and covered by a peel caused by the harmful effect of the amniotic fluid. Babies born with gastroschisis suffer from gastrointestinal dysmotility. The present aim was to verify whether the myenteric plexus is damaged in a rat model of gastroschisis. In the gastroschisis rat model fetus, the myenteric plexus was not yet organized in the well-defined ganglia and, in the most damaged loops, the neuronal cells were scattered or absent. Immunohistochemistry for alpha-internexin and peripherin (markers of neuronal maturity) gave results similar to those of earlier embryonic ages. These findings indicate a delay in neuronal differentiation and myenteric plexus organization that might play a role in the postnatal dysmotility observed in gastroschisis.

Computer-assisted stereology: point fraction of lung parenchyma and alveolar surface density in fetal and newborn sheep.

The aim of this study was to determine the accuracy of a new computer-assisted stereological technique in obtaining structural information of the lung. We compared the point fraction of lung parenchyma (Pp) and alveolar surface density (Sv) obtained by established manual point/intercept counting methods and compared them with those obtained using a computer-assisted method. Lung tissues obtained from normally grown fetal sheep (n = 6) and from newborn lambs with severe lung hypoplasia (n = 5) were inflation fixed via the trachea and processed for light microscopy. In verification-of-technique experiments, Pp and Sv correlated well with known values. There was a significant linear correlation between manual and computer-assisted stereological measurements for values of Pp (r2 = 0.92) and Sv (r2 = 0.98). Our data lead us to believe that the computer-assisted stereological technique described in this study provides accurate estimates of Pp and Sv and hence may be a valuable tool for evaluating the effects of factors upon structural development of the lung.

Pulmonary epithelial liquid absorption, expressed in relation to alveolar surface area, is reduced in fetal lambs following in utero tracheal occlusion.

We examined the effect of accelerated lung growth, induced by in utero tracheal occlusion (TO), on lung liquid uptake in near-term fetal sheep. In utero TO was performed in five fetal sheep at 110 days of gestation (term, approximately 145 days); six SHAM operated fetuses served as controls. The rate of liquid movement across the pulmonary epithelium was measured, using a previously established technique, in anesthetized fetal sheep between 133-137 days of gestation during a 2-hr adrenaline infusion (0.50 microg/min/kg, I.V.) and while lung luminal pressure was maintained at 5 mmHg. The rate of fetal lung liquid uptake was linear in all fetuses (mean r(2) < 0.97, n = 11). Mean values of lung liquid uptake expressed in relation to dry lung weight and luminal surface area of the right lung were significantly lower in TO fetuses (1.8 +/- 0.3 mL/hr/g and 1.0 +/- 0.2 mL/hr/m(2)) than in SHAM fetuses (2.6 +/- 0.2 mL/hr/g and 1.8 +/- 0.1 mL/hr/m(2)); surface area of the right lung was 140% greater in TO fetuses than in SHAM fetuses. There was a linear relationship between lung liquid uptake and pulmonary epithelial surface area in SHAM animals, but not in TO fetuses. We hypothesize that loss of alveolar epithelial type-II cells induced by increased levels of fetal lung expansion may impair alveolar liquid clearance in the perinatal period.

In utero hematopoietic stem cell therapy.

In utero hematopoietic stem cell transplantation (IUHSCTx) is a promising approach for the treatment of a potentially large number of fetuses affected by congenital hematologic disorders. With technical advances in prenatal diagnosis and fetal intervention, the majority of these diseases can now be diagnosed early in gestation, allowing consideration of prenatal treatment. It, therefore, stands to reason that there is increasing interest in performing in utero hematopoietic stem cell transplantation at many centers around the world. Although the approach remains experimentally promising, expansion of clinical application will depend on improved understanding of the biological barriers to engraftment in the fetus as well as on the development of effective clinical strategies based on the hematopoietic biology of individual disorders.

Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males.

A potential consequence of systemic administration of viral vectors is the inadvertent introduction of foreign DNA into recipient germ cells. To evaluate the safety of in vivo recombinant adeno-associated virus (rAAV) mediated gene transfer approaches for hemophilia B, we explored the risk of germline transmission of vector sequences following intramuscular (IM) injection of rAAV in four species of male animals (mouse, rat, rabbit and dog). In vector biodistribution studies in mice and rats, there is a dose-dependent increase in the likelihood that vector sequences can be detected in gonadal DNA using a sensitive PCR technique. However, in dogs DNA extracted from semen is negative for vector sequences. To address this discrepancy, studies were done in rabbits, and both semen and testicular DNAs were analyzed for the presence of vector sequences. These studies showed that no AAV vector sequences were detected in DNA extracted from rabbit semen samples collected at time points ranging from 7 to 90 days following IM injection of 1 x 10(13) vector genomes rAAV (vg) per kg. In contrast, DNA extracted from gonadal tissue was positive for vector sequences, but the positive signals diminished in number and strength with time. By FISH analysis, AAV signals were localized to the testis basement membrane and the interstitial space; no intracellular signal was observed. We observed similar findings following hepatic artery administration of rAAV in rats and dogs, suggesting that our findings are independent of the route of administration of vector. Attempts to transduce isolated murine spermatogonia directly with AAV-lacZ were unsuccessful. In clinical studies human subjects injected IM with an AAV vector at doses up to 2 x 10(12) vg/kg have shown no evidence of vector sequences in semen. Together, these studies suggest that rAAV introduced into skeletal muscle or the hepatic artery does not transduce male germ cells efficiently. We conclude that the risk of inadvertent germline transmission of vector sequences following IM or hepatic artery injection of AAV-2 vectors is extremely low.