Polyinosinic acid blocks adeno-associated virus macrophage endocytosis in vitro and enhances adeno-associated virus liver directed gene therapy in vivo

2013 ◽  
pp. 130815223935004
Author(s):  
Remco van Dijk ◽  
Paula Montenegro-Miranda ◽  
Christel Riviere ◽  
Ronald Schilderink ◽  
Lysbeth ten Bloemendaal ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Adeno Associated Virus ◽  
Polyinosinic Acid

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 Identification and Validation of Small Molecules That Enhance Recombinant Adeno-associated Virus Transduction following High-Throughput Screens

2016 ◽  
Vol 90 (16) ◽  
pp. 7019-7031 ◽  
Author(s):  
Sarah C. Nicolson ◽  
Chengwen Li ◽  
Matthew L. Hirsch ◽  
Vincent Setola ◽  
R. Jude Samulski
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
High Throughput ◽  
Adeno Associated Virus ◽  
Virus Gene ◽  
Diploid Cells ◽  
High Throughput Screens ◽  
Group 1

ABSTRACTWhile the recent success of adeno-associated virus (AAV)-mediated gene therapy in clinical trials is promising, challenges still face the widespread applicability of recombinant AAV(rAAV). A major goal is to enhance the transduction efficiency of vectors in order to achieve therapeutic levels of gene expression at a vector dose that is below the immunological response threshold. In an attempt to identify novel compounds that enhance rAAV transduction, we performed two high-throughput screens comprising 2,396 compounds. We identified 13 compounds that were capable of enhancing transduction, of which 12 demonstrated vector-specific effects and 1 could also enhance vector-independent transgene expression. Many of these compounds had similar properties and could be categorized into five groups: epipodophyllotoxins (group 1), inducers of DNA damage (group 2), effectors of epigenetic modification (group 3), anthracyclines (group 4), and proteasome inhibitors (group 5). We optimized dosing for the identified compounds in several immortalized human cell lines as well as normal diploid cells. We found that the group 1 epipodophyllotoxins (teniposide and etoposide) consistently produced the greatest transduction enhancement. We also explored transduction enhancement among single-stranded, self-complementary, and fragment vectors and found that the compounds could impact fragmented rAAV2 transduction to an even greater extent than single-stranded vectors.In vivoanalysis of rAAV2 and all of the clinically relevant compounds revealed that, consistent with ourin vitroresults, teniposide exhibited the greatest level of transduction enhancement. Finally, we explored the capability of teniposide to enhance transduction of fragment vectorsin vivousing an AAV8 capsid that is known to exhibit robust liver tropism. Consistent with ourin vitroresults, teniposide coadministration greatly enhanced fragmented rAAV8 transduction at 48 h and 8 days. This study provides a foundation based on the rAAV small-molecule screen methodology, which is ideally used for more-diverse libraries of compounds that can be tested for potentiating rAAV transduction.IMPORTANCEThis study seeks to enhance the capability of adeno-associated viral vectors for therapeutic gene delivery applicable to the treatment of diverse diseases. To do this, a comprehensive panel of FDA-approved drugs were tested in human cells and in animal models to determine if they increased adeno-associated virus gene delivery. The results demonstrate that particular groups of drugs enhance adeno-associated virus gene delivery by unknown mechanisms. In particular, the enhancement of gene delivery was approximately 50 to 100 times better with than without teniposide, a compound that is also used as chemotherapy for cancer. Collectively, these results highlight the potential for FDA-approved drug enhancement of adeno-associated virus gene therapy, which could result in safe and effective treatments for diverse acquired or genetic diseases.

Incorporation of the factor IX Padua mutation into FIX-Triple improves clotting activity in vitro and in vivo

2013 ◽  
Vol 110 (08) ◽  
pp. 244-256 ◽  
Author(s):  
Chung-Yang Kao ◽  
Shu-Jhu Yang ◽  
Mi-Hua Tao ◽  
Yung-Ming Jeng ◽  
I-Shing Yu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Replacement Therapy ◽  
Therapeutic Potential ◽  
Factor Ix ◽  
Adeno Associated Virus ◽  
Viral Dose ◽  
Better Than

SummaryUsing gain-of-function factor IX (FIX) for replacement therapy for haemophilia B (HB) is an attractive strategy. We previously reported a high-activity FIX, FIX-Triple (FIX-V86A/E277A/R338A) as a good substitute for FIX-WT (wild-type) in protein replacement therapy, gene therapy, and cell therapy. Here we generated a new recombinant FIXTripleL (FIX-V86A/E277A/R338L) by replacing the alanine at residue 338 of FIX-Triple with leucine as in FIX-Padua (FIX-R338L). Purified FIX-TripleL exhibited 22-fold higher specific clotting activity and 15-fold increased binding affinity to activated FVIII compared to FIXWT. FIX-TripleL increased the therapeutic potential of FIX-Triple by nearly 100% as demonstrated with calibrated automated thrombogram and thromboelastography. FIX-TripleL demonstrated a normal clearance rate in HB mice. The clotting activity of FIX-TripleL was consistently 2- to 3-fold higher in these mice than that of FIX-Triple or FIXR338L. Gene delivery of adeno-associated virus (AAV) in HB mice showed that FIX-TripleL had 15-fold higher specific clotting activity than FIX-WT, and this activity was significantly better than FIX-Triple (10-fold) or FIX-R338L (6-fold). At a lower viral dose, FIX-TripleL improved FIX activity from sub-therapeutic to therapeutic levels. Under physiological conditions, no signs of adverse thrombotic events were observed in long-term AAV-FIX-treated C57Bl/6 mice. Hepatocellular adenomas were observed in the high- but not the medium- or the lowdose AAV-treated mice expressing FIX-WT or FIX-Triple, indicating the advantages of using hyperfunctional FIX variants to reduce viral doses while maintaining therapeutic clotting activity. Thus, incorporation of the FIX Padua mutation significantly improves the clotting function of FIX-Triple so as to optimise protein replacement therapy and 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 Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages

2008 ◽  
Vol 89 (5) ◽  
pp. 1097-1105 ◽  
Author(s):  
Hidde J. Haisma ◽  
Jan A. A. M. Kamps ◽  
Gera K. Kamps ◽  
Josee A. Plantinga ◽  
Marianne G. Rots ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Kupffer Cells ◽  
Transgene Expression ◽  
Liver Toxicity ◽  
Viral Vector ◽  
Adenovirus Vector ◽  
Liver Macrophages ◽  
Polyinosinic Acid

Adenovirus is among the preferred vectors for gene therapy because of its superior in vivo gene-transfer efficiency. However, upon systemic administration, adenovirus is preferentially sequestered by the liver, resulting in reduced adenovirus-mediated transgene expression in targeted tissues. In the liver, Kupffer cells are responsible for adenovirus degradation and contribute to the inflammatory response. As scavenger receptors present on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated the possible implication of these receptors in the clearance of the adenovirus vector. Polyinosinic acid [poly(I)], a scavenger receptor A ligand, was analysed for its capability to inhibit adenovirus uptake specifically in macrophages. In in vitro studies, the addition of poly(I) before virus infection resulted in a specific inhibition of adenovirus-induced gene expression in a J774 macrophage cell line and in primary Kupffer cells. In in vivo experiments, pre-administration of poly(I) caused a 10-fold transient increase in the number of adenovirus particles circulating in the blood. As a consequence, transgene expression levels measured in different tissues were enhanced (by 5- to 15-fold) compared with those in animals that did not receive poly(I). Finally, necrosis of Kupffer cells, which normally occurs as a consequence of systemic adenovirus administration, was prevented by the use of poly(I). No toxicity, as measured by liver-enzyme levels, was observed after poly(I) treatment. From our data, we conclude that poly(I) can prevent adenovirus sequestration by liver macrophages. These results imply that, by inhibiting adenovirus uptake by Kupffer cells, it is possible to reduce the dose of the viral vector to diminish the liver-toxicity effect and to improve the level of transgene expression in target tissues. In systemic gene-therapy applications, this will have great impact on the development of targeted adenoviral vectors.

Adeno‐associated virus 8‐mediated gene therapy for choroideremia: preclinical studies in in vitro and in vivo models

2014 ◽  
Vol 16 (5-6) ◽  
pp. 122-130 ◽  
Author(s):  
Aaron Black ◽  
Vidyullatha Vasireddy ◽  
Daniel C. Chung ◽  
Albert M. Maguire ◽  
Rajashekhar Gaddameedi ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Preclinical Studies ◽  
In Vivo Models ◽  
Adeno Associated Virus

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 An Alternate Route for Adeno-associated Virus (AAV) Entry Independent of AAV Receptor

2018 ◽  
Vol 92 (7) ◽  
Author(s):  
Amanda M. Dudek ◽  
Sirika Pillay ◽  
Andreas S. Puschnik ◽  
Claude M. Nagamine ◽  
Fang Cheng ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Viral Vector ◽  
Cell Attachment ◽  
Vector System ◽  
Large Set ◽  
Adeno Associated Virus ◽  
Systemic Injection ◽  
Entry Pathway

ABSTRACTDeterminants and mechanisms of cell attachment and entry steer adeno-associated virus (AAV) in its utility as a gene therapy vector. Thus far, a systematic assessment of how diverse AAV serotypes engage their proteinaceous receptor AAVR (KIAA0319L) to establish transduction has been lacking, despite potential implications for cell and tissue tropism. Here, a large set of human and simian AAVs as well asin silico-reconstructed ancestral AAV capsids were interrogated for AAVR usage. We identified a distinct AAV capsid lineage comprised of AAV4 and AAVrh32.33 that can bind and transduce cells in the absence of AAVR, independent of the multiplicity of infection. Virus overlay assays and rescue experiments in nonpermissive cells demonstrate that these AAVs are unable to bind to or use the AAVR protein for entry. Further evidence for a distinct entry pathway was observedin vivo, as AAVR knockout mice were equally as permissive to transduction by AAVrh32.33 as wild-type mice upon systemic injection. We interestingly observe that some AAV capsids undergo a low level of transduction in the absence of AAVR, bothin vitroandin vivo, suggesting that some capsids may have a multimodal entry pathway. In aggregate, our results demonstrate that AAVR usage is conserved among all primate AAVs except for those of the AAV4 lineage, and a non-AAVR pathway may be available to other serotypes. This work furthers our understanding of the entry of AAV, a vector system of broad utility in gene therapy.IMPORTANCEAdeno-associated virus (AAV) is a nonpathogenic virus that is used as a vehicle for gene delivery. Here, we have identified several situations in which transduction is retained in both cell lines and a mouse model in the absence of a previously defined entry receptor, AAVR. Defining the molecular determinants of the infectious pathway of this highly relevant viral vector system can help refine future applications and therapies with this vector.

scholarly journals Transduction of Renal Cells in Vitro and in Vivo by Adeno-Associated Virus Gene Therapy Vectors

1999 ◽  
Vol 10 (9) ◽  
pp. 1908-1915 ◽  
Author(s):  
MICHAEL S. LIPKOWITZ ◽  
BASIL HANSS ◽  
NATALIE TULCHIN ◽  
PATRICIA D. WILSON ◽  
JESSICA C. LANGER ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Gene Delivery ◽  
Mesangial Cells ◽  
Renal Diseases ◽  
Primary Cells ◽  
Renal Cells ◽  
Adeno Associated Virus

Abstract. There has been an increasing interest recently in the possibility of treating renal diseases using gene therapy. The ability to pursue gene therapy for renal diseases has been limited by the availability of an adequate system for gene delivery to the kidney. Adeno-associated virus (AAV) is a defective virus of the parvovirus family that has a number of properties attractive for renal gene delivery: recombinant AAV contains no viral genes; expression of genes delivered by these vectors does not activate cell-mediated immunity; the virus is able to transduce nondividing as well as dividing cells; and both wild-type and recombinant AAV integrate into the host chromosome resulting in long-term gene expression. Studies were performed to determine whether AAV can deliver reporter genes to kidney cells in vitro and in vivo. These studies show that AAV can deliver reporter genes with approximately equal efficiency to human mesangial, proximal tubule, thick ascending limb, collecting tubule, and renal cell carcinoma cells in primary culture. Immortalized mouse mesangial cells are transduced at a much greater efficiency. Transduction can be enhanced by pharmaceutical agents up to sevenfold in primary cells (transducing up to 20% of primary cells per well) and as much as 400-fold in immortalized mesangial cells. AAV delivered in vivo by intraparenchymal injection results in at least 3 mo of reporter gene expression in tubular epithelial, but not glomerular or vascular, cells at the injection site. These data indicate that AAV can deliver genes to renal cells both in vitro and in vivo resulting in prolonged gene expression, and thus AAV can be a useful tool for renal gene delivery.

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