scholarly journals Enhanced Transgene Expression from Recombinant Single-Stranded D-Sequence-Substituted Adeno-Associated Virus Vectors in Human Cell LinesIn Vitroand in Murine HepatocytesIn Vivo

2014 ◽  
Vol 89 (2) ◽  
pp. 952-961 ◽  
Author(s):  
Chen Ling ◽  
Yuan Wang ◽  
Yuan Lu ◽  
Lina Wang ◽  
Giridhara R. Jayandharan ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Human Cell ◽  
Transgene Expression ◽  
Human Gene ◽  
Adeno Associated Virus ◽  
Human Virus ◽  
Human Gene Therapy ◽  
Virus Serotype

ABSTRACTWe have previously reported that the removal of a 20-nucleotide sequence, termed the D sequence, from both ends of the inverted terminal repeats (ITRs) in the adeno-associated virus serotype 2 (AAV2) genome significantly impairs rescue, replication, and encapsidation of the viral genomes (X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Mol Biol 250:573–580, 1995; X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Virol 70:1668–1677, 1996). Here we describe that replacement of only one D sequence in either ITR restores each of these functions, but DNA strands of only single polarity are encapsidated in mature progeny virions. Since most commonly used recombinant AAV vectors contain a single-stranded DNA (ssDNA), which is transcriptionally inactive, efficient transgene expression from AAV vectors is dependent upon viral second-strand DNA synthesis. We have also identified a transcription suppressor sequence in one of the D sequences, which shares homology with the binding site for the cellular NF-κB-repressing factor (NRF). The removal of this D sequence from, and replacement with a sequence containing putative binding sites for transcription factors in, single-stranded AAV (ssAAV) vectors significantly augments transgene expression both in human cell linesin vitroand in murine hepatocytesin vivo. The development of these genome-modified ssAAV vectors has implications not only for the basic biology of AAV but also for the optimal use of these vectors in human gene therapy.IMPORTANCEThe results of the studies described here not only have provided novel insights into some of the critical steps in the life cycle of a human virus, the adeno-associated virus (AAV), that causes no known disease but have also led to the development of novel recombinant AAV vectors which are more efficient in allowing increased levels of gene expression. Thus, these studies have significant implications for the potential use of these novel AAV vectors in human gene therapy.

scholarly journals Adeno-Associated Virus Type 2-Mediated Gene Transfer: Correlation of Tyrosine Phosphorylation of the Cellular Single-Stranded D Sequence-Binding Protein with Transgene Expression in Human Cells In Vitro and Murine Tissues In Vivo

1998 ◽  
Vol 72 (2) ◽  
pp. 1593-1599 ◽  
Author(s):  
Keyun Qing ◽  
Benjawan Khuntirat ◽  
Cathryn Mah ◽  
Dagmar M. Kube ◽  
Xu-Shan Wang ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Dna Synthesis ◽  
Progenitor Cells ◽  
Transgene Expression ◽  
Human Gene ◽  
Adeno Associated Virus ◽  
Human Gene Therapy

ABSTRACT Although the adeno-associated virus type 2 (AAV)-based vector system has gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy, the single-stranded nature of the viral genome, and consequently the rate-limiting second-strand viral DNA synthesis, significantly affect its transduction efficiency. We have identified a cellular tyrosine phosphoprotein, designated the single-stranded D sequence-binding protein (ssD-BP), which interacts specifically with the D sequence at the 3′ end of the AAV genome and may prevent viral second-strand DNA synthesis in HeLa cells (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879–10884, 1997). In the present studies, we examined whether the phosphorylation state of the ssD-BP correlates with the ability of AAV to transduce various established and primary cells in vitro and murine tissues in vivo. The efficiencies of transduction of established human cells by a recombinant AAV vector containing the β-galactosidase reporter gene were 293 > KB > HeLa, which did not correlate with the levels of AAV infectivity. However, the amounts of dephosphorylated ssD-BP which interacted with the minus-strand D probe were also as follows: 293 > KB > HeLa. Predominantly the phosphorylated form of the ssD-BP was detected in cells of the K562 line, a human erythroleukemia cell line, and in CD34+ primary human hematopoietic progenitor cells; consequently, the efficiencies of AAV-mediated transgene expression were significantly lower in these cells. Murine Sca-1+ lin − primary hematopoietic stem/progenitor cells contained predominantly the dephosphorylated form of the ssD-BP, and these cells could be efficiently transduced by AAV vectors. Dephosphorylation of the ssD-BP also correlated with expression of the adenovirus E4orf6 protein, known to induce AAV gene expression. A deletion mutation in the E4orf6 gene resulted in a failure to catalyze dephosphorylation of the ssD-BP. Extracts prepared from mouse brain, heart, liver, lung, and skeletal-muscle tissues, all of which are known to be highly permissive for AAV-mediated transgene expression, contained predominantly the dephosphorylated form of the ssD-BP. Thus, the efficiency of transduction by AAV vectors correlates well with the extent of the dephosphorylation state of the ssD-BP in vitro as well as in vivo. These data suggest that further studies on the cellular gene that encodes the ssD-BP may promote the successful use of AAV vectors in human gene therapy.

scholarly journals Adeno-Associated Virus Type 2-Mediated Gene Transfer: Role of Cellular FKBP52 Protein in Transgene Expression

2001 ◽  
Vol 75 (19) ◽  
pp. 8968-8976 ◽  
Author(s):  
Keyun Qing ◽  
Jonathan Hansen ◽  
Kirsten A. Weigel-Kelley ◽  
Mengqun Tan ◽  
Shangzhen Zhou ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Dna Synthesis ◽  
Transgene Expression ◽  
Human Gene ◽  
Transduction Efficiency ◽  
Adeno Associated Virus ◽  
Human Gene Therapy

ABSTRACT Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful vector for human gene therapy, the transduction efficiencies of AAV vectors vary greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays a crucial role in AAV-mediated transgene expression (K. Y. Qing, X.-S. Wang, D. M. Kube, S. Ponnazhagan, A. Bajpai, and A. Srivastava, Proc. Natl. Acad. Sci. USA 94:10879–10884, 1997). We have documented a strong correlation between the phosphorylation state of ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing, B. Khuntrirat, C. Mah, D. M. Kube, X.-S. Wang, S. Ponnazhagan, S. Z. Zhou, V. J. Dwarki, M. C. Yoder, and A. Srivastava, J. Virol. 72:1593–1599, 1998). We have also established that the ssD-BP is phosphorylated by epidermal growth factor receptor protein tyrosine kinase and that the tyrosine-phosphorylated form, but not the dephosphorylated form, of ssD-BP prevents AAV second-strand DNA synthesis and, consequently, results in a significant inhibition of AAV-mediated transgene expression (C. Mah, K. Y. Qing, B. Khuntrirat, S. Ponnazhagan, X.-S. Wang, D. M. Kube, M. C. Yoder, and A. Srivastava, J. Virol. 72:9835–9841, 1998). Here, we report that a partial amino acid sequence of ssD-BP purified from HeLa cells is identical to a portion of a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein 52 (FKBP52). FKBP52 was purified by using a prokaryotic expression plasmid containing the human cDNA. The purified protein could be phosphorylated at both tyrosine and serine or threonine residues, and only the phosphorylated forms of FKBP52 were shown to interact with the AAV single-stranded D-sequence probe. Furthermore, in in vitro DNA replication assays, tyrosine-phosphorylated FKBP52 inhibited AAV second-strand DNA synthesis by greater than 90%. Serine- or threonine-phosphorylated FKBP52 caused ≈40% inhibition, whereas dephosphorylated FKBP52 had no effect on AAV second-strand DNA synthesis. Deliberate overexpression of FKBP52 effectively reduced the extent of tyrosine phosphorylation of the protein, resulting in a significant increase in AAV-mediated transgene expression in human and murine cell lines. These studies corroborate the idea that the phosphorylation status of the cellular FKBP52 protein correlates strongly with AAV transduction efficiency, which may have important implications for the optimal use of AAV vectors in human gene therapy.

scholarly journals The 37/67-Kilodalton Laminin Receptor Is a Receptor for Adeno-Associated Virus Serotypes 8, 2, 3, and 9

2006 ◽  
Vol 80 (19) ◽  
pp. 9831-9836 ◽  
Author(s):  
Bassel Akache ◽  
Dirk Grimm ◽  
Kusum Pandey ◽  
Stephen R. Yant ◽  
Hui Xu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Cultured Cells ◽  
Human Gene ◽  
Laminin Receptor ◽  
Transduction Efficiency ◽  
Adeno Associated Virus ◽  
Virus Serotype ◽  
Tumor Gene

ABSTRACT Adeno-associated virus serotype 8 (AAV8) is currently emerging as a powerful gene transfer vector, owing to its capability to efficiently transduce many different tissues in vivo. While this is believed to be in part due to its ability to uncoat more readily than other AAV serotypes such as AAV2, understanding all the processes behind AAV8 transduction is important for its application and optimal use in human gene therapy. Here, we provide the first report of a cellular receptor for AAV8, the 37/67-kDa laminin receptor (LamR). We document binding of LamR to AAV8 capsid proteins and intact virions in vitro and demonstrate its contribution to AAV8 transduction of cultured cells and mouse liver in vivo. We also show that LamR plays a role in transduction by three other closely related serotypes (AAV2, -3, and -9). Sequence and deletion analysis allowed us to map LamR binding to two protein subdomains predicted to be exposed on the AAV capsid exterior. Use of LamR, which is constitutively expressed in many clinically relevant tissues and is overexpressed in numerous cancers, provides a molecular explanation for AAV8's broad tissue tropism. Along with its robust transduction efficiency, our findings support the continued development of AAV8-based vectors for clinical applications in humans, especially for tumor gene therapy.

scholarly journals Genome Editing Technologies: From Bench Side to Bedside

Acta Medica ◽  
2018 ◽  
Vol 49 (3) ◽  
pp. 30
Author(s):  
Fulya Yaylacıoğlu Tuncay ◽  
Pervin Rukiye Dinçer
Keyword(s):  
Gene Therapy ◽  
Animal Models ◽  
Genome Editing ◽  
Clinical Studies ◽  
Human Gene ◽  
Human Gene Therapy ◽  
Novel Technologies ◽  
Preclinical And Clinical Studies

The development of genome editing technologies has given the chance to researchers to manipulate any genomic sequences precisely. This ability is very useful for creating animal models to study human diseases in vivo; for easy creation of isogenic cell lines to study in vitro and most importantly for overcoming many disadvantages that the researchers faced during the human gene therapy trials. Here we review the basic mechanisms of genome editing technology and the four genome-editing platforms. We also discuss the applications of these novel technologies in preclinical and clinical studies in four groups according to the mechanism used, and lastly, summarize the problems in these technologies.

Adeno-associated virus vectors for human gene therapy

Gene Therapy ◽  
2020 ◽  
pp. 77-92
Author(s):  
Jeffrey S. Bartlett ◽  
Richard J. Samulski
Keyword(s):  
Gene Therapy ◽  
Human Gene ◽  
Adeno Associated Virus ◽  
Human Gene Therapy ◽  
Virus Vectors
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