scholarly journals Structure of Adeno-Associated Virus Vector DNA following Transduction of the Skeletal Muscle

1999 ◽  
Vol 73 (3) ◽  
pp. 1949-1955 ◽  
Author(s):  
Nathalie Vincent-Lacaze ◽  
Richard O. Snyder ◽  
Régis Gluzman ◽  
Delphine Bohl ◽  
Catherine Lagarde ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Gene Transfer ◽  
Transgene Expression ◽  
De Novo ◽  
Early Time ◽  
Progressive Increase ◽  
Stable Gene ◽  
Adeno Associated Virus ◽  
Rolling Circle ◽  
Time Points

ABSTRACT The skeletal muscle provides a very permissive physiological environment for adeno-associated virus (AAV) type 2-mediated gene transfer. We have studied the early steps leading to the establishment of permanent transgene expression, after injection of recombinant AAV (rAAV) particles in the quadriceps muscle of mice. The animals received an rAAV encoding a secreted protein, murine erythropoietin (mEpo), under the control of the human cytomegalovirus major immediate-early promoter and were sacrificed between 1 and 60 days after injection. The measurement of plasma Epo levels and of hematocrits indicated a progressive increase of transgene expression over the first 2 weeks, followed by a stabilization at maximal plateau values. The rAAV sequences were analyzed by Southern blotting following neutral or alkaline gel electrophoresis of total DNA from injected muscles. While a high number of rAAV sequences were detected during the first 5 days following the injection, only a few percent of these sequences was retained in the animals analyzed after 2 weeks, in which transgene expression was maximal. Double-stranded DNA molecules resulting from de novo second-strand synthesis were detected as early as day 1, indicating that this crucial step of AAV-mediated gene transfer is readily accomplished in the muscle. The templates driving stable gene expression at later time points are low in copy number and structured as high-molecular-weight concatemers or interlocked circles. The presence of the circular form of the rAAV genomes at early time points suggests that the molecular transformations involved in the formation of stable concatemers may involve a rolling-circle type of DNA replication.

Systemic AAV9.LAMP2B injection reverses metabolic and physiologic multiorgan dysfunction in a murine model of Danon disease

2020 ◽  
Vol 12 (535) ◽  
pp. eaax1744 ◽  
Author(s):  
Ana Maria Manso ◽  
Sherin I. Hashem ◽  
Bradley C. Nelson ◽  
Emily Gault ◽  
Angel Soto-Hermida ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Gene Transfer ◽  
Murine Model ◽  
Autophagic Flux ◽  
Adeno Associated Virus ◽  
Multiorgan Dysfunction ◽  
Danon Disease ◽  
Physiologic Function ◽  
Male Patients ◽  
Dose Dependent

Danon disease (DD) is a rare X-linked autophagic vacuolar myopathy associated with multiorgan dysfunction, including the heart, skeletal muscle, and liver. There are no specific treatments, and most male patients die from advanced heart failure during the second or third decade of life. DD is caused by mutations in the lysosomal-associated membrane protein 2 (LAMP2) gene, a key mediator of autophagy. LAMP2 has three isoforms: LAMP2A, LAMP2B, and LAMP2C. LAMP2B is the predominant isoform expressed in cardiomyocytes. This study evaluates the efficacy of human LAMP2B gene transfer using a recombinant adeno-associated virus 9 carrying human LAMP2B (AAV9.LAMP2B) in a Lamp2 knockout (KO) mouse, a DD model. AAV9.LAMP2B was intravenously injected into 2- and 6-month-old Lamp2 KO male mice to assess efficacy in adolescent and adult phenotypes. Lamp2 KO mice receiving AAV9.LAMP2B demonstrated dose-dependent restoration of human LAMP2B protein in the heart, liver, and skeletal muscle tissue. Impaired autophagic flux, evidenced by increased LC3-II, was abrogated by LAMP2B gene transfer in all tissues in both cohorts. Cardiac function was also improved, and transaminases were reduced in AAV9.LAMP2B-treated KO mice, indicating favorable effects on the heart and liver. Survival was also higher in the older cohort receiving high vector doses. No anti-LAMP2 antibodies were detected in mice that received AAV9.LAMP2B. In summary, LAMP2B gene transfer improves metabolic and physiologic function in a DD murine model, suggesting that a similar therapeutic approach may be effective for treating patients with this highly morbid disease.

One year transgene expression with adeno-associated virus cardiac gene transfer

2005 ◽  
Vol 100 (3) ◽  
pp. 421-426 ◽  
Author(s):  
Y. Joseph Woo ◽  
Janet C.L. Zhang ◽  
Matthew D. Taylor ◽  
Jeffrey E. Cohen ◽  
Vivian M. Hsu ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Transgene Expression ◽  
Adeno Associated Virus ◽  
Cardiac Gene ◽  
One Year

Stable Gene Transfer and Expression in Human Primary T-Cells by the Sleeping Beauty Transposon System.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5539-5539
Author(s):  
Xianzheng Zhou ◽  
Xin Huang ◽  
Andrew C. Wilber ◽  
Lei Bao ◽  
Dong Tuong ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Sleeping Beauty ◽  
Stable Gene ◽  
Cell Clones ◽  
Stable Gene Expression

Abstract The Sleeping Beauty (SB) transposon system is a non-viral DNA delivery system in which a transposase directs integration of an SB transposon into TA-dinucleotide sites in the genome. To determine whether the SB transposon system can mediate integration and long-term transgene expression in human primary T-cells, freshly isolated peripheral blood lymphocytes (PBLs) without prior activation were nucleofected with SB vectors carrying a DsRed reporter gene. Plasmids containing the SB transposase on the same (cis) (n=10) or separate molecule (trans) (n=8) as the SB transposon mediated long-term and stable reporter gene expression in human primary T-cells. We observed that delivery of SB transposase-encoding plasmid in trans effectively mediated stable gene expression in primary T-cells, exhibiting about a 3-fold increase (11% vs. 3% with 10 microgram plasmid on day 21) in potency in comparison with the cis vector (p<0.0001). In addition, a transposase mutant construct was incapable of mediating stable gene expression in human PBLs (n=6, p<0.0001), confirming that catalytic DDE domain is necessary for transposition in human primary T-cells. Immunophenotyping analysis in transposed T-cells showed that both CD4 and CD8 T-cells were transgene positive. SB-mediated high level of transgene expression in human T-cells was maintained in culture for at least 4 months without losing observable expression. Southern hybridization analysis showed a variety of transposon integrants among the 6 DsRed positive T-cell clones and no transposon sequences identifiable in the 2 DsRed negative clones. Sequencing of transposon:chromosome junctions in 5 out of 6 transposed T-cell clones confirmed that stable gene expression was due to SB-mediated transposition. In other studies, PBLs were successfully transfected using the SB transposon system and shown to stably and functionally express a fusion protein consisting of a surface receptor useful for positive T-cell selection and a “suicide” gene useful for elimination of transfected T-cells after chemotherapy. This study is the first report demonstrating that the SB transposon system can mediate stable gene transfer in human primary PBLs, which may be more advantageous for T-cell based gene therapies over widely used virus-based or conventional mammalian DNA vectors in terms of simplicity, stability, efficiency and safety.

scholarly journals Adeno-Associated Virus Serotype 6 Capsid Tyrosine-to-Phenylalanine Mutations Improve Gene Transfer to Skeletal Muscle

2010 ◽  
Vol 21 (10) ◽  
pp. 1343-1348 ◽  
Author(s):  
Chunping Qiao ◽  
Wei Zhang ◽  
Zhenhua Yuan ◽  
Jin-Hong Shin ◽  
Jianbin Li ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Gene Transfer ◽  
Adeno Associated Virus ◽  
Virus Serotype

scholarly journals Muscle-Specific Overexpression of the Adenovirus Primary Receptor CAR Overcomes Low Efficiency of Gene Transfer to Mature Skeletal Muscle

2001 ◽  
Vol 75 (9) ◽  
pp. 4276-4282 ◽  
Author(s):  
J. Nalbantoglu ◽  
N. Larochelle ◽  
E. Wolf ◽  
G. Karpati ◽  
H. Lochmuller ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Gene Transfer ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Tibialis Anterior Muscle ◽  
Gene Promoter ◽  
Fold Increase ◽  
Developmentally Regulated ◽  
Mouse Muscle ◽  
Low Efficiency

ABSTRACT Significant levels of adenovirus (Ad)-mediated gene transfer occur only in immature muscle or in regenerating muscle, indicating that a developmentally regulated event plays a major role in limiting transgene expression in mature skeletal muscle. We have previously shown that in developing mouse muscle, expression of the primary Ad receptor CAR is severely downregulated during muscle maturation. To evaluate how global expression of CAR throughout muscle affects Ad vector (AdV)-mediated gene transfer into mature skeletal muscle, we produced transgenic mice that express the CAR cDNA under the control of the muscle-specific creatine kinase promoter. Five-month-old transgenic mice were compared to their nontransgenic littermates for their susceptibility to AdV transduction. In CAR transgenics that had been injected in the tibialis anterior muscle with AdVCMVlacZ, increased gene transfer was demonstrated by the increase in the number of transduced muscle fibers (433 ± 121 in transgenic mice versus 8 ± 4 in nontransgenic littermates) as well as the 25-fold increase in overall β-galactosidase activity. Even when the reporter gene was driven by a more efficient promoter (the cytomegalovirus enhancer–chicken β-actin gene promoter), differential transducibility was still evident (893 ± 149 versus 153 ± 30 fibers; P < 0.001). Furthermore, a fivefold decrease in the titer of injected AdV still resulted in significant transduction of muscle (253 ± 130 versus 14 ± 4 fibers). The dramatic enhancement in AdV-mediated gene transfer to mature skeletal muscle that is observed in the CAR transgenics indicates that prior modulation of the level of CAR expression can overcome the poor AdV transducibility of mature skeletal muscle and significant transduction can be obtained at low titers of AdV.

Stabilized HIF‐1α is superior to VEGF for angiogenesis in skeletal muscle via adeno‐associated virus gene transfer

2005 ◽  
Vol 19 (10) ◽  
pp. 1365-1367 ◽  
Author(s):  
Katri Pajusola ◽  
Jaana Künnapuu ◽  
Sanna Vuorikoski ◽  
Jarkko Soronen ◽  
Helder André ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Gene Transfer ◽  
Adeno Associated Virus ◽  
Virus Gene

scholarly journals T Cell-Mediated Immune Responses to AAV and AAV Vectors

2021 ◽  
Vol 12 ◽  
Author(s):  
Hildegund C. J. Ertl
Keyword(s):  
T Cell ◽  
Gene Transfer ◽  
Immune Responses ◽  
De Novo ◽  
T Cell Responses ◽  
Adeno Associated Virus ◽  
Effector Functions ◽  
Cell Responses ◽  
High Doses

Adeno-associated virus (AAV)-mediated gene transfer has benefited patients with inherited diseases, such as hemophilia B, by achieving long-term expression of the therapeutic transgene. Nevertheless, challenges remain due to rejection of AAV-transduced cells, which in some, but not all, patients can be prevented by immunosuppression. It is assumed that CD8+ T cells induced by natural infections with AAVs are recalled by the AAV vector’s capsid and upon activation eliminate cells expressing the degraded capsid antigens. Alternatively, it is feasible that AAV vectors, especially if given at high doses, induce de novo capsid- or transgene product-specific T cell responses. This chapter discusses CD8+ T cell responses to AAV infections and AAV gene transfer and avenues to prevent their activation or block their effector functions.

scholarly journals Leishmania infection induces a limited differential gene expression in the sand fly midgut

2020 ◽  
Author(s):  
Iliano V. Coutinho-Abreu ◽  
Tiago D. Serafim ◽  
Claudio Meneses ◽  
Shaden Kamhawi ◽  
Fabiano Oliveira ◽  
...  
Keyword(s):  
Gene Expression ◽  
Developmental Stages ◽  
De Novo ◽  
Early Time ◽  
Differentially Expressed ◽  
Sand Fly ◽  
Leishmania Infection ◽  
Lutzomyia Longipalpis ◽  
Late Time ◽  
Time Points

Abstract Background: Phlebotomine sand flies are the vectors of Leishmania worldwide. To develop in the sand fly midgut, Leishmania multiplies and undergoes multiple stage differentiations leading to the infective form, the metacyclic promastigotes. To gain a better understanding of the influence of Leishmania infection on midgut gene expression, we performed RNA-Seq comparing uninfected and Leishmania infantum -infected Lutzomyia longipalpis midguts at seven time points which cover the various Leishmania developmental stages including early time points when blood digestion is taking place, and late time points when the parasites are undergoing metacyclogenesis. Results: Out of over 13,841 transcripts assembled de novo , only 113 sand fly transcripts, about 1%, were differentially expressed. Further, we observed a low overlap of differentially expressed sand fly transcripts across different time points suggesting that each Leishmania stage influences midgut gene expression in a specific manner. Two main patterns of sand fly gene expression modulation were noted. At early time points (days 1-4), more transcripts were down-regulated by Leishmania infection at large fold changes (> -32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 4 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by small fold changes (< 32 fold). The molecular function of these genes has been associated with the metabolism of lipids and detoxification of xenobiotics. Conclusion: Overall, it appears that Leishmania modulates sand fly gene expression early on in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points, where a massive number of parasites in the anterior midgut results only in modest changes in midgut gene expression.

scholarly journals Leishmania infection induces a limited differential gene expression in the sand fly midgut

2019 ◽  
Author(s):  
Iliano V. Coutinho-Abreu ◽  
Tiago D. Serafim ◽  
Claudio Meneses ◽  
Shaden Kamhawi ◽  
Fabiano Oliveira ◽  
...  
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Early Time ◽  
Differentially Expressed ◽  
Sand Fly ◽  
Leishmania Infection ◽  
Lutzomyia Longipalpis ◽  
Late Time ◽  
Blood Digestion ◽  
Time Points

Abstract Background: Phlebotomine sand flies are the vectors of Leishmania worldwide. To develop in the sand fly midgut, Leishmania multiplies and undergoes multiple stage differentiations leading to the infective form, the metacyclic promastigotes. To gain a better understanding of the influence of Leishmania infection in midgut gene expression, we performed RNA-Seq comparing uninfected Lutzomyia longipalpis midguts and Leishmania infantum-infected Lutzomyia longipalpis midguts at seven time points which cover the various developmental Leishmania stages including early time points when blood digestion is taking place and late time points when the parasites are undergoing metacyclogenesis. Results: Out of over 13,841 transcripts assembled de novo, only 113 sand fly transcripts, about 1%, were differentially expressed. Further, we observed a low overlap of differentially expressed sand fly transcripts across different time points suggesting a specific influence of each Leishmania stage on midgut gene expression. Two main patterns of sand fly gene expression modulation were noticed. At early time points (days 1-4), more transcripts were down-regulated by Leishmania infection at large fold changes (> -32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 4 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by small fold changes (< 32 fold), and the molecular function of such genes are associated with the metabolism of lipids and detoxification of xenobiotics (P450). Conclusion: Overall, it appears that Leishmania modulates sand fly gene expression early on in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points, when modest midgut gene expression changes correlate with a massive amount of parasites in the anterior midgut.

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