Gene therapy in neuromuscular disorders / Terapia gênica nas doenças neuromusculares

Abstract Monogenic neuromuscular disorders are potentially treatable through gene therapy. Using viral vectors, a therapeutic transgene aims to restore normal levels of a protein not produced by the defective gene, or to silence a gene whose expression leads to toxic effects. Spinal Muscular Atrophy (SMA) is a good example of a monogenic disease that currently has an AAV9-based vector gene therapy as a therapeutic option. In this review, we intend to discuss the viral vectors and their mechanisms of action, in addition to reviewing the clinical trials that supported the approval of gene therapy (AVXS-101) for SMA as well as neuromuscular diseases that are potentially treatable with gene replacement therapy.

Resumo Doenças neuromusculares monogênicas são potencialmente tratáveis através de terapia gênica. Utilizando-se de vetores virais, um transgene terapêutico objetiva repor os níveis normais de uma proteina não produzida pelo gene defeituoso ou silenciar um gene cuja expressão leva a efeitos tóxicos. A Atrofia Muscular Espinhal (AME) é um bom exemplo de doença monogenica que atualmente tem uma terapia gênica com vetor viral AAV9 como opção terapêutica. Nesta revisão, pretendemos discutir os vetores virais e macanismos de ação utilizados, além de revisar os ensaios clínicos que embasaram a aprovação da terapia gênica (AVXS-101) para AME, assim como doenças neuromusculares potencialmente tratáveis com terapia de reposição gênica.

AAV-Mediated Astrocyte-Specific Gene Expression under Human ALDH1L1 Promoter in Mouse Thalamus

Adeno-associated virus (AAV)-mediated gene delivery has been proposed to be an essential tool of gene therapy for various brain diseases. Among several cell types in the brain, astrocyte has become a promising therapeutic target for brain diseases, as more and more contribution of astrocytes in pathophysiology has been revealed. Until now, genetically targeting astrocytes has been possible by utilizing the glial fibrillary acidic protein (GFAP) promoter. In some brain areas including thalamus, however, the GFAP expression in astrocytes is reported to be low, making it difficult to genetically target astrocytes using GFAP promoter. To study the function of astrocytes in thalamus, which serves as a relay station, there is a great need for identifying an alternative astrocyte-specific promoter in thalamus. Recently, a new astrocyte-specific promoter of ALDH1L1 has been identified. However, it has not been examined in thalamus. Here we developed and characterized an AAV vector expressing Cre recombinase under the human ALDH1L1 promoter, AAV-hALDH1L1-Cre. To test the cell-type specific expression of AAV-hALDH1L1-Cre, AAV virus was injected into several brain regions of Ai14 (RCL-tdTomato) mouse, which reports Cre activity by tdTomato expression. In thalamus, we observed that tdTomato was found mostly in astrocytes (91.71%), with minimal occurrence in neurons (2.67%). In contrast, tdTomato signal was observed in both neurons and astrocytes of the amygdala (neuron: 68.13%, astrocyte: 28.35%) and hippocampus (neuron: 76.25%, astrocyte: 18.00%), which is consistent with the previous report showing neuronal gene expression under rat ALDH1L1 promoter. Unexpectedly, tdTomato was found mostly in neurons (91.98%) with minimal occurrence in astrocytes (6.66%) of the medial prefrontal cortex. In conclusion, hALDH1L1 promoter shows astrocyte-specificity in thalamus and may prove to be useful for targeting thalamic astrocytes in mouse.

Repair effects of co-expression of the VEGF and BMP genes via an adeno-associated viral vector on early steroid-in-duced avascular necrosis of the femoral head in rabbits / 中华骨科杂志

Objective To investigate the repair effects of co-expression of the VEGF and BMP genes via an adeno-as-sociated viral vector on early steroid-induced avascular necrosis of the femoral head in rabbits. Methords To construct ani-mal model of early SANFH and screen by MRI. The SANFH animal were divided into rAAV-IRES-hrGFP(AAV-GFP), rAAV-hVEGF165-IRES-hrGFP(AAV-VEGF), rAAV-hBMP-7-IRES-hrGFP(AAV-BMP)and rAAV-hVEGF165-IRES-hBMP-7(AAV-VEGF/BMP)groups. The four group virus vectors were injected into core decompression region at the dose of 25μl/site after core decompression operation directly. Repair effects of rAAV vector on early SANFH in rabbits were evaluated by Western blot assay, HE staining, immunohistochemical staining, MRI, radionuclide bone scan, blood vessel counting detected by ink perfusion and fro-zen section, Micro-CT and biomechanical strength detection on the 12th week post-injection. Results Model success ratio was 73.33%. rAAV-hVEGF165-IRES-hBMP-7 virus vector efficiently expressed hVEGF165 and hBMP-7 genes on the 12th week after rAAV injection. hVEGF165 protein secreted in vivo promoted metabolism in core decompression region by increasing the quantity of new vessels and improving the blood supply;hBMP-7 protein secreted in vivo promoted new bone formation in core decompres-sion region by increasing bone mineral density and improving bone biomechanical strength. The AAV-VEGF/BMP group can pro-mote repair effects more effectively than AAV-VEGF group or AAV-BMP group. Conclusion The adeno-associated viral vectors co-expressing hVEGF165 and hBMP-7 can promote repair effects on early SANFH in rabbits by increasing the blood supply and strengthening the bone quality of femoral head.

Combination therapy of adeno-associated virus-mediated expression of vascular endothelial growth factor gene and bone marrow mesenchymal stem cells for limb ischemia / 中华老年医学杂志

Objective To evaluate the angiogenic effect of the bone marrow mesenchymal stem cells (MSCs) transfected with adeno-associated virus (rAAV) -mediated human vascular endothelial growth factor 165 (VEGF165) gene, to detect the expression and bioactivity of VEGF in the MSCs.and to detect the expression and bioactivity of VEGF165 gene in the area of ischemic skeletal muscle in rats. Methods The MSCs were cultured by whole bone marrow culture method, then the MSCs were transfected with rAAV-VEGF165, the expression of VEGF was examined using ELISA and RT-PCR. The model of ischemic skeletal muscle was established by ligation in inbred rats. The rats were injected with PBS at the ischemic zone (group A), MSC (group B), hVEGF-transfected MSC (group D), and 7 days later they were injected with hVEGF transfected MSC (group C). Six weeks after the transfection, the capillary density of the ischemic zone was examined by factor Ⅷ stain. Results The MSCs were cultured successfully according to the expressions of positive CD44 and CD90,negative CD34. In rAVV-VEGF165 transfected group, the secretion levels of VEGF165 in supernatant were significantly higher than in non-transfected group 1,3,5,7 and 9 days after injection [(131. 98±6.00) ng/L vs. (68. 72±1.99) ng/L; (263. 96±4.58) ng/L vs. (76. 47±4.98) ng/L;(540. 85±5.97) ng/L vs. (89. 86± 1.99) ng/L; (208. 98± 5.06) ng/L vs. (84. 93±8. 97) ng/L;(174.45±5.00) ng/L vs. (68.71±5.98) ng/L, all P＜0.05]. On the position of 579 bp, highbrightness strip could be seen. There were no significant differences in growth curve and cell morphology between transfected group and non-transfected group. Six weeks after the transplantation, the capillary density was significantly greater in group D [(9.35 ± 2.72)/ vision]than in group A [(1. 05±0. 50)/vision] ,group B [(3.10± 1.43)/vision, both P＜0. 01)] and group C [(6. 95± 1.69)/ vision, P＜0. 05] Conclusions MSCs are helpful for the stable expression of hVEGF gene, and it is good cellular vehicle for VEGF genes. The effect of combined therapy is much better than separate transplantation of MSCs. The best time to transplant is 10 days after surgical operation.

Protective Effect of Recombinant Adeno-Associated Virus 2/8-Mediated Gene Therapy from the Maternal Hyperphenylalaninemia in Offsprings of a Mouse Model of Phenylketonuria

Phenylketonuria (PKU) is an autosomal recessively inherited metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH). The accumulation of phenylalanine leads to severe mental and psychomotor retardation, and the fetus of an uncontrolled pregnant female patient presents with maternal PKU syndrome. We have reported previously on the cognitive outcome of biochemical and phenotypic reversal of PKU in a mouse model, Pah(enu2), by the AAV serotype 2-mediated gene delivery of a human PAH transgene. However, the therapeutic efficacy had been limited to only male PKU mice. In this study, we generated a pseudotyped recombinant AAV2/8-hPAH vector and infused it into female PKU mice through the hepatic portal vein or tail vein. Two weeks after injection, complete fur color change to black was observed in female PKU, as in males. The PAH activity in the liver increased to 65-70% of the wild-type activity in female PKU mice and to 90% in male PKU mice. Plasma phenylalanine concentration in female PKU mice decreased to the normal value. In addition, the offsprings of the treated female PKU mice can rescue from the harmful effect of maternal hyperphenylalaninemia. These results indicate that recombinant AAV2/8-mediated gene therapy is a potential therapeutic strategy for PKU.

Promoter effects of adeno-associated viral vector for transgene expression in the cochlea in vivo

The aims of this study were to evaluate the expression of enhanced green fluorescent protein (EGFP) driven by 6 different promoters, including cytomegalovirus IE enhancer and chicken beta-actin promoter (CAG), cytomegalovirus promoter (CMV), neuron-specific enolase promoter (NSE), myosin 7A promoter (Myo), elongation factor 1alpha promoter (EF-1alpha), and Rous sarcoma virus promoter (RSV), and assess the dose response of CAG promoter to transgene expression in the cochlea. Serotype 1 adeno-associated virus (AAV1) vectors with various constructs were transduced into the cochleae, and the level of EGFP expression was examined. We found the highest EGFP expression in the inner hair cells and other cochlear cells when CAG promoter was used. The CMV and NSE promoter drove the higher EGFP expression, but only a marginal activity was observed in EF-1alpha promoter driven constructs. RSV promoter failed to driven the EGFP expression. Myo promoter driven EGFP was exclusively expressed in the inner hair cells of the cochlea. When driven by CAG promoter, reporter gene expression was detected in inner hair cells at a dose as low as 3 x 10(7) genome copies, and continued to increase in a dose- dependent manner. Our data showed that individual promoter has different ability to drive reporter gene expression in the cochlear cells. Our results might provide important information with regard to the role of promoters in regulating transgene expression and for the proper design of vectors for gene expression and gene therapy.

A novel way of therapeutic angiogenesis using an adeno-associated virus-mediated angiogenin gene transfer

To develop a novel therapeutic angiogenesis for the treatment of cardiovascular diseases, angiogenin (ANG1) was examined as a potential therapeutic gene. An adeno-associated virus (AAV)-mediated gene delivery system was used to measure the therapeutic efficacy of ANG1. Using a triple co-transfection technique, rAAV-ANG1-GFP, rAAV- VEGF-GFP and rAAV-GFP vectors were produced, which were then used to infect human umbilical vein endothelial cells (HUVECs) in order to evaluate in vitro angiogenic activities. Their protein expressions, tagged with green fluorescent protein (GFP), were monitored by confocal microscopy. The functional activities were measured using wound-healing HUVEC migration assays. The number of migrated cells stimulated by both the expressed ANG1 and the VEGF in rAAV-infected HUVECs increased almost twice the number observed in the expressed GFP control. In vivo angiogenic activities of the expressed ANG1 or VEGF were determined using mouse angiogenesis assays. The angiogenic activities of ANG1 or VEGF expressed in the injected mice were increased by 1.36 and 2.16 times, respectively, compared to those of the expressed GFP control. These results demonstrate that the expressed ANG1 derived from rAAV infection has in vitro and in vivo angiogenic activities and suggest that the rAAV-ANG1 vector is a potential strategy for therapeutic angiogenesis.

Low-dose radiation response of the p21(WAF1/CIP1) gene promoter transduced by adeno-associated virus vector

In cancer gene therapy, restriction of antitumor transgene expression in a radiation field by use of ionizing radiation-inducible promoters is one of the promising approaches for tumor-specific gene delivery. Although tumor suppressor protein p53 is induced by low doses (<1 Gy) of radiation, there have been only a few reports indicating potential utilization of a p53-target gene promoter, such as that of the p21 gene. This is mainly because the transiently transfected promoter of p53-target genes is not much sensitive to radiation. We examined the response of the p21 gene promoter to low-dose radiation when transduced into a human breast cancer cell line MCF-7 by use of recombinant adeno-associated virus (rAAV) vectors. It was shown that the p21 gene promoter transduced by rAAV vectors was more highly radiation-responsive than that transiently transfected by electroporation. A significant induction of the p21 gene promoter by radiation of low doses down to 0.2 Gy was observed. When cells were transduced with the p21 gene promoter-driven HSVtk gene by rAAV vector, they were significantly sensitized to repetitive treatment with low dose radiation (1 Gy) in the presence of the prodrug ganciclovir. It was therefore considered that the p21 gene promoter in combination with a rAAV vector is potentially usable for the development of a low-dose radiation-inducible vector for cancer gene therapy.

Recombinant adeno-associated virus mediated gene transfer in a mouse model for homocystinuria

Homocystinuria is a metabolic disorder caused by a deficiency of cystathionine b-synthase (CBS). The major clinical symptoms of this disease are mental retardation, lens dislocation, vascular disease with life-threatening thromboembolisms, and skeletal deformities. The major treatments for CBS deficiency include pharmacologic doses of pyridoxine or dietary restriction of methionine. There is currently no effective long-term treatment to lower the elevated plasma levels of homocysteine. However, gene therapy could be an effective novel approach for the treatment of homocystinuria. A recombinant adeno- associated virus vector carrying human CBS cDNA (rAAV-hCBS) was constructed and administered to CBS-/- mice by intramuscular (IM) and intraperitoneal (IP) injections. Serum homocysteine concentrations significantly decreased in treated mice compared with age-matched controls two weeks after treatment. The treated CBS-/- mice had life spans 3-7 days longer compared with untreated CBS-/- mice. In CBS-/- mice treated with rAAV-hCBS via IP injection, the vector was detected in all organs examined including liver, spleen, and kidney, and CBS gene expression was observed by immunohistochemical staining in the liver. These results indicate the efficacy of gene delivery and demonstrate the possibility of gene therapy mediated by AAV gene transfer in this mouse model of homocystinuria.

Construction of recombinant adeno-associated virus vector expressing human tissue kallikrein gene and its effect on human umbilical vein endothelial cells / 中华老年医学杂志

Objective To construct the recombinant adeno-associated virus vector(rAAV) expressing the human tissue kallikrein(HK)gene and to detect the expression of interested gene in human umbilical vein endothelial cells(HUVEC)which were infected with different titers of rAAV. Methods The HK gene was cloned into the pAAV-MCS and co-transfected AAV-293 cells with other two plasmids(the pAAV-RC,and pHelper)by lipofectamine.The recombinant AAV(rAAV HK)particles was harvested and its viral titer was measured.HUVEC were infected with different titers of rAAV-HK particles.Seventy-two hours later,the expression of the interested gene was detected by RT-PCR and ELISA.Results The AAV expression system of human tissue kallikrein gene was successfully constructed.The viral titer of recombinant AAV reached 6.2?10~7 particles/ml. When compared with the control group,the transcription of HK gene in the group which was infected with rAAV-HK increased significantly[(0.59?0.12)vs(0.26?0.05)(P＜0.05)],and the expression of HK in the HUVEC was three times more than that in the control[(120.1?40.9)vs (30.8?12.8)](P＜0.001).The transcription of eNOSmRNA in the infected HUVEC was higher than that of the control[(1.19?0.28)vs(0.66?0.11)](P＜0.05).The transcription of caspase-3 mRNA was lower than that of the control[(0.30?0.25)vs(0.67?0.27)](P＜0.05).And there was no obvious variation happened in the transcription of VEGF,ET-1,TR-B,bradykinin B1 receptor and Bradykinin B2 receptor.Conclusions When co-transfected AAV-293 cells with three plasmids (pAAV-HK,pAAV-RC,pHelper),the HK gene expression is significantly and stably increased. Introducing HK gene into HUVEC can improve endothelial transfection efficiency.

Induction of neovascularization in pigs by recombinant adeno-associated virus 2 vector encoding human VEGF_(165 ) cDNA / 中国介入心脏病学杂志

Objective To investigate the effect of rAAV2 VEGF 165 (recombinant adeno associated Virus 2 vector encoding VEGF 165 cDNA) in stimulating angiogenesis, its dose effect relationship in swine acute coronary occlusion model, and to search for a suitable dose for further investigation in swine myocardium ischemia model Methods Ligation of LAD (left anterior descending coronary artery) was performed in swine modles via mini thoracotomy rAAV2 VEGF 165 (experimental group) or phosphate buffer saline (control group) was injected around the infarcted region The experimental group was divided into four subgroups: according to the amount of virus genome, which were: 1?10 11 v g (virus genome), 5?10 11 v g, 1?10 12 v g and 5?10 12 v g Animals underwent angiographic evaluation of collateral development four weeks after the operation Expression of VEGF was assessed by semiquantitative RT PCR and immunohistochemical staining Results Four weeks after the operation, expression of VEGF was detected in all the four experimental subgroups and the level of expression was higher than that of the control group Capillary density was higher in both of the 1?10 12 v g and 5?10 12 v g subgroups, compared with the control Coronary angiography demonstrated higher collateral index to the occluded LAD in the groups of 5?10 11 v g,1?10 12 v g and 5?10 12 v g than that of the control Conclusion Direct injection of rAAV2 VEGF 165 can achieve effective expression of VEGF in swine acute ischemic myocardium At certain dosage, rAAV2 VEGF 165 can promote generation of blood ressels in swine acute ischemic myocardium and 1? 10 12 v g is a more suitable dosage for swine myocardium ischemia model

Long-term behavioral recovery in Parkinsonian rats by triple transduction with dopamine-biosynthesizing enzyme genes / 中华神经科杂志

Objective To observe the effect of gene therapy for Parkinson disease by triple transduction with dopamine-biosynthesizing enzyme genes. Methods We constructed high titer purified adeno-associated virus (AAV) vectors containing cDNAs encoding human tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC) and GTP cyclohydrolase I (GCH) respectively. AAV-TH, AAV-AADC and AAV-GCH were stereotaxically injected into the denervated striatum of Parkinsonian rat; the expression of TH, AADC or GCH was assayed by using immunohistochemical staining; the rotational behavior was assayed for 1 year.Results The immunohistochemical staining and behavioral testing showed that all of TH, AADC and GCH were expressed stably in the striatum, and the rats treated with AAV-TH, AAV-AADC and AAV-GCH showed more remarkable decrease in rotation rate persisted for at least 1 year, than those treated with AAV-TH and AAV-AADC(P

Biological effects of adeno-associated virus vectors of human transforming growth factor ?_1 and ?_3 for reversion of rabbit intervertebral disc degeneration / 中华骨科杂志

Objective To study the biological effects of pSNAV2-hTGF?1 and pSNAV2-hTGF?3 on the reversion of rabbit disc degeneration. Methods Rabbit nucleus pulpous and annulus fibrosus cells were isolated and cultured. The fluorescence labled pSNAV2 were used to detect the transfect rates of rabbit disc cells at first. Then, the pSNAV2-hTGF?1 and pSNAV2-hTGF?3 were transfected into the degenerated rabbit disc cells respectively. The biological effects of hTGF?1 and hTGF?3 on degenerated rabbit disc cells were detected with Western-bloting and 35S detection to analyze and compare the matrix synthesis of the tranfected cells. Results pSNAV2 could transfect degenerated disc cells effectively in the early stages. Both the pSNAV2-hTGF?1 and pSNAV2-hTGF?3 could stimulate the synthesis of collagen Ⅱ and proteoglycan of the rabbit disc cells. For the early stage of degenerated disc cells, the synthesis of collagen Ⅱ and proteoglycan were greater transfected with pSNAV2-hTGF?1 than transfected with pSNAV2-hTGF?3. The pSNAV2-hTGF?1 could promote the degenerated rabbit annulus fibrosus cells to synthesize collagen Ⅰ and pSNAV2-hTGF?3 could promote the degenerated nucleus pulpous cells of later stage to synthesize the collagen Ⅱ. Conclusion Both pSNAV2-hTGF?1 and pSNAV2-hTGF?3 can promote the degenerated rabbit disc cells of early stage to synthesize the matrix. pSNAV2-hTGF?3 can efficently promote the seriously degenerated nucleus pulpous cells to synthesize the collagen Ⅱ.