Prenatal Gene Research Articles

Stem cells and the frontiers of neonatology

The aim of the most recent studies on regenerative medicine was to focus on capability of stem cells deriving not only from haematopoietic system, but also from other organ and tissues, to regenerate damaged tissues. Stem cells derived from foetal annexes such as cord blood, placenta and amniotic fluid can be currently used in the effort to treat prenatally diagnosed genetic diseases. Cells derived from cord blood have been used since 1988 as an alternative source to realize stem cell transplantation. Compared with bone marrow, cord blood has shown the advantages of quick availability, less risk of GHVD, together with higher compatibility rates, and less risk of infections. Mesenchymal stem cells (MSCs) are multi-potent stem cells able to differentiate into different lineages, including osteocytes, chondrocytes, and adipocytes. Because of their trafficking capacity to injured tissues, clinical trials have been started evaluating the use of MSCs in the treatment of metabolic diseases like Hurler syndrome and metachromatic leukodystrophy, or Osteogenesis Imperfecta. MSCs were initially identified in adult bone marrow (BM-MSC), but cells resembling BM-MSCs have also been found in other tissues, both adult (peripheral blood, synovial membrane) and foetal (peripheral blood, liver, spleen, placenta, umbilical cord, and amniotic membrane). BM-MSCs have been widely used in clinical applications, as for cell-based therapy of Osteogenesis Imperfecta and metabolic diseases. In addition, human multi-potent MSCs present in second-trimester amniotic fluids may be a good target for prenatal gene therapy because of their expandability, their ability to differentiate into multiple lineages and their high transduction efficiency.

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.

Gene diagnosis for spinal muscular atrophy and its application study

To establish an effective testing system for gene diagnosis, carrier detection and prenatal diagnosis for spinal muscular atrophy (SMA). Twenty-six patients with SMA were directly tested with PCR-RFLP for exon 7 deletion in the SMN1 gene. Carrier detection was carried out with multi-PCR-DHPLC. Amniotic fluid was taken at the middle stage of gestation from pregnant women who had given birth to affected children. Twenty-five out of 26 patients were diagnosed as having SMN1 gene deletion. Fifty-two of their parents were found to be carriers of exon 7 deletion. Eight of 20 fetuses were diagnosed as having SMN1 gene deletion by PCR-RFLP. PCR-RFLP and multi-PCR-DHPLC techniques can provide rapid diagnosis for exon 7 deletion detection and carrier detection. PCR-RFLP may also be adapted for prenatal gene diagnosis of exon 7 deletion in SMN1 gene.

Germ Line Gene Transmission in Prenatal Gene Therapy – An Approach to Ethical Judgement

Article Germ Line Gene Transmission in Prenatal Gene Therapy – An Approach to Ethical Judgement was published on April 1, 2009 in the journal Journal of international biotechnology law (volume 6, issue 2).

Genetic Testing in the Diagnostic Evaluation of Inherited Platelet Disorders

Inherited disorders of platelets give rise to rare bleeding syndromes through defects of platelet function and/or platelet production. Platelet function testing by biological and immunologic assays can identify the loss or abnormal functioning of specific receptor systems, signaling pathways, storage organelles, or enzymatic activities essential for adhesion, activation, and aggregation. In vitro culture of megakaryocytes can help identify the origin of familial thrombocytopenias, and electron microscopy can point to ultrastructural defects and giant platelet syndromes. But a full diagnosis can only be complete when the genetic defect has been defined for each patient. Glanzmann thrombasthenia (GT) and the Bernard-Soulier syndrome are the most studied of the membrane disorders and a wide range of mutations identified. We will use GT as an example to show how genetic studies can help understand the cell biology, pathophysiology, and management of a group of rare diseases where many of the genetic causes remain to be elucidated. Knowledge of the mutation in GT and whether it affects either of the ITGA2B or ITGB3 genes will be essential as we enter the period where accurate prenatal diagnosis and gene therapy may become viable options.

Correction of ADAMTS13 Deficiency by In Utero Gene Transfer of Lentiviral Vector encoding ADAMTS13 Genes

Deficiency of A Disintegrin And Metalloprotease with ThromboSpondin (ADAMTS13) results in thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective treatment to date. We show in this study that an administration of a self-inactivating lentiviral vector encoding human full-length ADAMTS13 and a variant truncated after the spacer domain (MDTCS) in mice by in utero injection at embryonic days 8 and 14 resulted in detectable plasma proteolytic activity (approximately 5-70%), which persisted for the length of the study (up to 24 weeks). Intravascular injection via a vitelline vein at E14 was associated with significantly lower rate of fetal loss than intra-amniotic injection, suggesting that the administration of vector at E14 may be a preferred gestational age for vector delivery. The mice expressing ADAMTS13 and MDTCS exhibited reduced sizes of von Willebrand factor (vWF) compared to the Adamts13(-/-) mice expressing enhanced green fluorescent protein (eGFP). Moreover, the mice expressing both ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time as compared to the Adamts13(-/-) expressing eGFP. The data demonstrate the successful correction of the prothrombotic phenotypes in Adamts13(-/-) mice by a single in utero injection of lentiviral vectors encoding human ADAMTS13 genes, providing the basis for developing a gene therapy for hereditary TTP in humans.

Prenatal gene therapy for the early treatment of genetic disorders

Prenatal gene therapy aims to deliver genes to cells and tissues early in prenatal life, allowing the correction of a genetic defect before long-term tissue damage has occurred. In contrast to postnatal gene therapy, prenatal application has a number of advantages, including targeting genes in a large population of dividing stem cells, and the smaller fetal size, which allows a higher vector-to-target cell ratio to be achieved. Early gestational delivery may result in the fetus developing immune tolerance to the transgenic protein, which would allow postnatal repeat of vector administration if needed. Recent advances in vector design and stem cell research have benefited this potential treatment. Although still in the preclinical stage, proof-of-principle studies in animal models of congenital disease, such as the hemophilic mouse, have shown the potential of prenatal gene therapy. Investigators have devised delivery strategies in large animals that could be used clinically to apply gene therapy to a human fetus, and prenatal gene therapy may be available for the treatment of certain life-threatening congenital disorders in the near future. With this treatment, pregnant women and their partners would have a third choice when faced with an affected fetus, where currently the only options are terminating the pregnancy or continuing with an affected fetus with a poor prognosis. A number of problems need to be resolved, including the long-term safety and efficacy of the therapy, the potential for germ-line gene transfer, how best to achieve informed consent for the therapy, as well as regulatory and ethical issues. In this article, we evaluate the current state and future implications of prenatal therapy in severe genetic diseases and outline the factors that will influence its movement into clinical practice.

Gene diagnosis for nine Chinese patients with DMD/BMD by multiplex ligation‐dependent probe amplification and prenatal diagnosis for one of them

This study aims to perform gene diagnosis for nine patients with Duchenne/Becker muscular dystrophy (DMD/BMD) and their parents with multiplex ligation-dependent probe amplification (MLPA), and to carry out prenatal gene diagnosis for one of them. Genomic DNA of the peripheral blood and fetal amniotic fluid cell was extracted from the pedigrees' members with DMD/BMD. Gene diagnosis was performed for theses pedigrees' members using a SALSA KIT. Short tandem repeats (STR) genotyping and X-linkage analysis were performed for the pedigree members of the fetus, which was used in the prenatal diagnosis. MLPA analysis results show that five of nine patients (DMD-1, DMD-2, DMD-4, DMD-8, and DMD-9) with DMD/BMD were found to have several hemizygous exon deletions in the dystrophin gene. The other patients and the fetus did not have any hemizygous deletion or duplication of any exons. The genomic DNA of the fetus was not contaminated by his mother's DNA as identified by STR genotyping. In addition, X-linkage analysis results show that the only X chromosome of the fetus comes from one of his mother's normal X chromosomes. Combined with STR genotyping and X-linkage analysis, MLPA is a convenient, highly effective and reliable gene diagnosis technique for congenital genetic disease.

Causes of errors in prenatal gene diagnosis and relevant countermeasures

产前诊断是一项高风险服务,疾病情况复杂、检测过程环节多,稍有疏漏就会出现差错.产前基因诊断有更强的针对性,不是通常意义上的确定胎儿是否异常,而是确定胎儿是否获得与先证者一样的基因型.本文就导致产前基因诊断差错的因素,从不同的环节给予分析。

Restricted transgene persistence after lentiviral vector‐mediated fetal gene transfer in the pregnant rabbit model

Prenatal gene transfer may enable early causal intervention for the treatment or prevention of many devastating diseases. Nevertheless, permanent correction of most inherited disorders requires a sustained level of expression from the therapeutic transgene, which could theoretically be achieved with integrating vectors. Rabbit fetuses received 8.5 x 10(6) HIV-based recombinant lentivirus particles containing the enhanced green fluorescent protein (EGFP) transgene by intrahepatic, intra-amniotic or intraperitoneal injection at 22 days of gestation. Provirus presence and transgene expression in rabbit tissues were evaluated at both 1.5 and 16 weeks post-in utero intervention by polymerase chain reaction (PCR) and reverse transcriptase-PCR, respectively. Moreover, we assessed persistence of EGFP by immunohistochemistry. Enzyme-linked immunosorbent assays confirmed the development of antibodies specific against both the viral vector and the reporter protein. Regardless of the route of administration employed, lentiviral vector-based in utero gene transfer was safe and reached 85% of the intervened fetuses at birth. However, the integrated provirus frequency was significantly reduced to 50% of that in young rabbits at 16 weeks post-treatment. In these animals, EGFP expression was evident in many tissues, including cytokeratin 5-rich basal cells from stratified and pseudostratified epithelia, suggesting that the lentiviral vector might have reached progenitor cells. Conversely, we identified the presence of immune-inflammatory infiltrates in several EGFP-expressing tissues. Moreover, almost 70% of the lentiviral vector-treated rabbits elicited a humoral immune response against the viral envelope and/or the EGFP. At two-thirds gestational age, the adaptive immune system of the rabbit appears a relevant factor limiting transgene persistence and expression following lentiviral vector-mediated in utero gene transfer.

Perinatal stem-cell and gene therapy for hemoglobinopathies

Most genetic diseases of the lymphohematopoietic system, including hemoglobinopathies, can now be diagnosed early in gestation. However, as yet, prenatal treatment is not available. Postnatal therapy by hematopoietic stem cell (HSC) transplantation from bone marrow, mobilized peripheral blood, or umbilical cord blood is possible for several of these diseases, in particular for the hemoglobinopathies, but is often limited by a lack of histocompatible donors, severe treatment-associated morbidity, and preexisting organ damage that developed before birth. In-utero transplantation of allogeneic HSC has been performed successfully in various animal models and recently in humans. However, the clinical success of this novel treatment is limited to diseases in which the fetus is affected by severe immunodeficiency. The lack of donor cell engraftment in nonimmunocompromised hosts is thought to be due to immunologic barriers, as well as to competitive fetal marrow population by host HSCs. Among the possible strategies to circumvent allogeneic HLA barriers, the use of gene therapy by genetically corrected autologous HSCs in the fetus is one of the most promising approaches. The recent development of strategies to overcome failure of efficient transduction of quiescent hematopoietic cells using new vector constructs and transduction protocols opens new perspectives for gene therapy in general, as well as for prenatal gene transfer in particular. The fetus might be especially susceptible for successful gene therapy approaches because of the developing, expanding hematopoietic system during gestation and the immunologic naiveté early in gestation, precluding immune reaction towards the transgene by inducing tolerance. Ethical issues, in particular regarding treatment safety, must be addressed more closely before clinical trials with fetal gene therapy in human pregnancies can be initiated.

Detection of a new mutation (T1140C) in a patient with Hunter syndrome from Guangdong, China

This study identified mutations of the idurnate-2-sulfatase (IDS) gene in a patient with Hunter syndrome, and established a basis for the diagnosis of the prenatal gene of Hunter syndrome. Urine glyeosaminoglycan (GAG) assay was used to make the preliminary diagnosis of mucopolysaccharidosis type II. Polymerase chain reaction (PCR) from dried blood spots and DNA sequencing were applied to analyze hotspot mutations in exons 9,3 and 8 of the IDS gene in the proband and his parents. A new missense mutation (T1140C) in exon 8 of the IDS gene was found by using DNA sequencing. This mutation caused a substitution of codon 339 from CTA (leucine) to CCA (praline). The patient is a hemizygote, and his mother is a heterozygote. The new missense mutation results in a change in the primary and tertiary structure of the IDS protein. It is possible that this mutation severely impairs enzymatic activity and is the underlying basis for the pathology seen in this patient with Hunter syndrome.

Distribution and expression of transgene green fluorescent protein in mice survived up to four weeks following in utero gene therapys

Gene replacement offers a potential cure for degenerative disorders caused by a single gene deletion or mutation. Diagnoses of monogenic disorders in the fetus enable prenatal gene replacement which may be beneficial from the perspectives of host inflammatory/immune response, efficacy and disease prevention. 
 Purpose: To evaluate the distribution and expression of reporter gene green fluorescent protein (GFP) in the tissues of mice survived up to one month following in utero gene therapy. 
 Methods: Vesicular Stomatitis Virus-G (VSV-G) pseudotyped lentiviral (LV) vector containing GFP was prepared via triple plasmid co-transfection. Time-mated CD-1 mice underwent individual amniotic sac injection with either 1x106 LV particles or saline (controls) on gestational day 16 (term=21d), and were allowed to undergo spontaneous parturition. Pups were sacrificed on postnatal days 0, 7, 21 and 28, and neonatal and maternal tissues were analyzed for GFP transgene (by DNA polymerase chain reaction; PCR), and transgene expression by quantitative reverse transcriptase (QRT) PCR and immunohistochemistry (IHC). 
 Results: We observed selective transduction of neonatal tissues (trachea, lung, liver, heart, kidney, spleen, intestine, skeletal muscle), in pups undergoing in utero transfection with LV-GFP. Maternal tissues did not contain transgene despite exposure during amniotic injection. Although the numbers of pups analyzed at each postnatal time point was small, we observed variable persistence of GFP expression that appeared to be tissue specific (with persistent expression noted in intestine of 4 week old pups). 
 Conclusions: Neonatal tissue transfection occurs in a variety of tissues following amniotic injection with LV-GFP in this murine model of in utero gene therapy. Transgene persistence and expression patterns observed over the first 4 weeks of life may reflect tissue-specific genomic insertion of transgene that favors persistent transcription in select tissues.

A new mutation (1062 del 16) of iduronate-2-sulfatase gene from a Chinese patient with hunter syndrome

To identify the mutations of iduronate-2-sulfatase (IDS) gene, to reveal its mutation features, and to establish a basis for genetic counseling and prenatal gene diagnosis of Hunter syndrome. Urine glycosaminoglycans (GAGs) assay, PCR and DNA sequencing were performed to detect mutation of IDS gene of the patient and his parents. The result showed that the patient was: DS(++), HS(++), KS(-), CS(-), and that both of his parents were negative. A frame-shift deletion mutation (1062 del 16) was identified in exon 7 of the patient's IDS gene. His parents' genotypes were normal. The patient's mutation was not inherited by his parents but a novel one. The mutation probably altered the primary structure and tertiary structure of IDS enzyme protein remarkably and lowered the activity of IDS enzyme greatly. Therefore it is supposed to be the direct cause of the disorder.

Gene Transfer to Ocular Stem Cells by Early Gestational Intraamniotic Injection of Lentiviral Vector

Ocular gene transfer has generally been approached by direct intraocular injection. In this study, we hypothesized that an opportunity exists during early gestation when specific ocular stem cell populations are accessible for gene transfer. These include the stem cell populations that maintain the cornea, lens, and retina throughout life. To test this hypothesis, we injected lentiviral vector encoding the green fluorescent protein (GFP) reporter gene into the murine amniotic space from the late head fold/early somite stage postcoital day 8 (E8) to E18 and performed sequential analysis of GFP expression in ocular tissues. Depending on the timing of vector exposure, significant GFP expression was observed in all ectoderm-derived tissues in the eye. With injection at early gestational time points, GFP expression persisted long term, with evidence of high efficiency stem cell transduction in the cornea, lens, and retina. The observed patterns and duration of gene expression confirm the accessibility of ocular stem cell populations for lentiviral vector-based gene transfer at specific developmental time points in early gestation. This model may be useful for the investigation of mechanisms of genetic and/or developmental ocular disease and for the development of prenatal gene therapy for specific ocular disorders.

Targeted Gene Transfer to Fetal Rat Lung Interstitium by Ultrasound-guided Intrapulmonary Injection

In utero gene transfer to the developing lung may have clinical or research applications. In this study, we developed a new method for specifically targeting the fetal rat lung with adeno and lentiviral vectors encoding the enhanced green fluorescence protein (EGFP) marker gene at E15.5 using ultrasound biomicroscopy (UBM). Survival rate, morphometric parameters, viral biodistribution, and lung transduction efficiency were analyzed and compared to the intra-amniotic route of administration. Expression of EGFP started as early as 24 and 72 h after the injection of adenoviral and lentiviral vectors, respectively. Both vectors transduced lung parenchyma with gene expression limited to interstitial cells of the injected region, in contrast to intra-amniotic injection, which targeted the pulmonary epithelium. Expression of EGFP was most intense at E18.5 and E21.5 for adenoviral and lentiviral vectors, respectively. In contrast to lentivirus, adenoviral expression significantly declined until final analysis at 1 week of age. This study demonstrates the feasibility of targeting the fetal rat lung interstitium with viral vectors under UBM guidance during the pseudoglandular stage. This model system may facilitate in vivo studies of dynamic lung morphogenesis and could provide insight into the efficacy of prenatal gene transfer strategies for treatment of specific lung disorders.

DISTRIBUTION AND EXPRESSION OF TRANSGENE GREEN FLUORESCENT PROTEIN IN MICE SURVIVING UP TO 4 WEEKS FOLLOWING IN UTERO GENE THERAPY.

Gene replacement offers a potential cure for degenerative disorders caused by a single gene deletion or mutation. Diagnoses of monogenic disorders in the fetus enable prenatal gene replacement, which may be beneficial from the perspectives of host inflammatory/immune response, efficacy, and disease prevention. Purpose The purpose of our study was to evaluate the distribution and expression of reporter gene green fluorescent protein (GFP) in the tissues of mice surviving up to 1 month following in utero gene therapy. Methods Used Vesicular stomatitis virus-G (VSV-G) pseudotyped lentiviral (LV) vector containing GFP was prepared via triple plasmid cotransfection. Time-mated CD-1 mice underwent individual amniotic sac injection with either 1 × 106 LV particles or saline (controls) on gestational day 16 (term = 21 days) and were allowed to undergo spontaneous parturition. Pups were sacrificed on postnatal days 0, 7, 21, and 28, and neonatal and maternal tissues were analyzed for GFP transgene (by DNA polymerase chain reaction; PCR) and transgene expression by quantitative reverse transcriptase (QRT) PCR and immunohistochemistry (IHC). Summary of Results We observed selective transduction of neonatal tissues (trachea, lung, liver, heart, kidney, spleen, intestine, skeletal muscle) in pups undergoing in utero transfection with LV-GFP. Maternal tissues did not contain transgene despite exposure during amniotic injection. Although the number of pups analyzed at each postnatal time point was small, we observed variable persistence of GFP expression that appeared to be tissue specific (with persistent expression noted in the intestine of 4-week-old pups). Conclusions Neonatal tissue transfection occurs in a variety of tissues following amniotic injection with LV-GFP in this murine model of in utero gene therapy. Transgene persistence and expression patterns observed over the first 4 weeks of life may reflect tissue-specific genomic insertion of transgene that favors persistent transcription in select tissues.

Evaluation of prenatal intra-amniotic LAMB3 gene delivery by adenoviral and AAV vectors in a mouse model of Herlitz junctional epidermolysis bullosa

Evaluation of prenatal intra-amniotic LAMB3 gene delivery by adenoviral and AAV vectors in a mouse model of Herlitz junctional epidermolysis bullosa

Evaluation of prenatal intra-amniotic LAMB3 gene delivery in a mouse model of Herlitz disease

Prenatal gene therapy has been considered for Herlitz junctional epidermolysis bullosa (H-JEB), a lethal genodermatosis caused by the absence of any of the three subunits of laminin-5, resulting from birth in widespread blistering and erosions of skin and mucosae. To investigate this strategy in an animal model, adenovirus type 5- and adeno-associated virus (AAV) type 2-derived vectors carrying a beta-galactosidase reporter gene or LAMB3 cDNA encoding the beta3 chain of laminin-5 were generated, tested for stability in amniotic fluid and evaluated in vitro on murine H-JEB keratinocytes, and in vivo by prenatal injection into the amniotic cavities of laminin-5 beta3-deficient mice. The different vectors were administered individually or combined at maximum doses on day 14 post coitum. Adenoviral vectors infected preferentially the foetal epidermis, whereas AAV delivered the transgene mainly to mucous membranes of the airways and the upper digestive tract. The LAMB3 transgene was expressed in target epithelia of newborn laminin-5 beta3-deficient mice, and the transgenic beta3 chain was shown to assemble with its endogenous partner chains, resulting in detectable amounts of laminin-5 in the basement membranes of skin and mucosae and in a lower extent of tissue separation in the skin. However, only combined delivery of the two vector types led to a minor increase of the life span of H-JEB mice. Failure to rescue diseased animals was, at least in part, due to abandonment of any conspicuous pup by the heterozygous mother. This is the first study of a prenatal gene therapy approach to a heritable blistering disorder. Although our findings indicate that prenatal combined administration of adenoviral and adeno-associated LAMB3 vectors provides therapeutic benefit to H-JEB mice, this animal model appears unsuitable for long-term investigations of the therapeutic concept.

Evaluation of prenatal intra-amniotic LAMB3 gene delivery by adenoviral and AAV vectors in a mouse model of Herlitz junctional epidermolysis bullosa

Background: Prenatal gene therapy has been considered for diseases such as Herlitz junctional epidermolysis bullosa (H-JEB), a lethal genodermatosis caused by absence of any of the three subunits of the basement membrane protein laminin-5. H-JEB presents from birth with widespread blistering of skin and mucosae and leads to death typically within the first year of life. In this study, we investigated in a mouse model whether H-JEB could be treated by gene delivery in utero.