Stability of Recombinant Mosaic Adeno-Associated Virus Vector rAAV/DJ/CAG at Different Temperature Conditions

2021 ◽  
Vol 17 (11) ◽  
pp. 2114-2124
Author(s):  
Alicja Bie´nkowska-Tokarczyk ◽  
Maciej Małecki
Keyword(s):  
Gene Therapy ◽  
Uv Light ◽  
Temperature Fluctuations ◽  
Effect Of Temperature ◽  
Transduction Efficiency ◽  
Microscopy Imaging ◽  
Adeno Associated Virus ◽  
Targeted Gene Delivery ◽  
Gene Therapy Vectors ◽  
The Stability

The nanometer size and biological characteristics of recombinant adeno-associated virus vectors (rAAV) make them particularly useful as gene therapy vectors and they have been successfully used in this role. Our latest research revealed that the rAAV/DJ/CAG mosaic vector offers highly efficient targeted gene delivery to melanoma cells metastasized to the lungs and that the transduction is temperature dependent. In order to further explore the ability of the rAAV/DJ/CAG vector to deliver highly selective transduction, this study was designed to identify the transduction stability of rAAV/DJ/CAG under various conditions. The temperatures used in this study ranged from −196 ° (liquid nitrogen) to 90 °, and the effect of temperature fluctuations (freeze-thaw, cooling-heating cycles) was also studied. This research also investigated the effects of UV radiation (ultraviolet) on the rAAV/DJ/CAG activity. Changes in the transduction efficiency were assessed via fluorescence microscopy imaging and the qPCR method. Under the test conditions, the transduction efficiency was reduced by approx. 35%, on average. High temperatures (70 °/90 °) and UV light proved to have the most detrimental impact. Changes in the stability of the rAAV/DJ/CAG structure are manifested by variations in the number of genome copies (gc) and GFP+ cells. Temperature fluctuations resulted in differences in the number of gc while maintaining a similar number of GFP+ cells, which may indicate specific changes in the rAAV/DJ/CAG structure, triggering disorders or degradation in the vector entry. This study provides interesting insights into rAAV/DJ/CAG, and the implications of these findings provide a basis for developing new protocols in cancer gene therapy.

scholarly journals Adeno-Associated Virus (AAV) Gene Delivery: Dissecting Molecular Interactions Upon Cell Entry

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1336
Author(s):  
Edward E. Large ◽  
Mark A. Silveria ◽  
Grant M. Zane ◽  
Onellah Weerakoon ◽  
Michael S. Chapman
Keyword(s):  
Gene Therapy ◽  
Molecular Interactions ◽  
Neutralizing Antibodies ◽  
Major Gene ◽  
Cell Entry ◽  
Transduction Efficiency ◽  
Adeno Associated Virus ◽  
Gene Therapy Vector ◽  
Gene Therapy Vectors ◽  
Factor Interactions

Human gene therapy has advanced from twentieth-century conception to twenty-first-century reality. The recombinant Adeno-Associated Virus (rAAV) is a major gene therapy vector. Research continues to improve rAAV safety and efficacy using a variety of AAV capsid modification strategies. Significant factors influencing rAAV transduction efficiency include neutralizing antibodies, attachment factor interactions and receptor binding. Advances in understanding the molecular interactions during rAAV cell entry combined with improved capsid modulation strategies will help guide the design and engineering of safer and more efficient rAAV gene therapy vectors.

scholarly journals Quantitation of Trace Levels of DNA Released from Disrupted Adeno-Associated Virus Gene Therapy Vectors

2021 ◽  
Author(s):  
Jared S. Bee ◽  
Kristin O'Berry ◽  
Yu (Zoe) Zhang ◽  
Megan Kuhn Phillippi ◽  
Akanksha Kaushal ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Adeno Associated Virus ◽  
Virus Gene ◽  
Gene Therapy Vectors

In Vivo Potency Assay for Adeno-Associated Virus–Based Gene Therapy Vectors Using AAVrh.10 as an Example

2018 ◽  
Vol 29 (3) ◽  
pp. 146-155 ◽  
Author(s):  
Bishnu P. De ◽  
Alvin Chen ◽  
Christiana O. Salami ◽  
Benjamin Van de Graaf ◽  
Jonathan B. Rosenberg ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Potency Assay ◽  
Adeno Associated Virus ◽  
Gene Therapy Vectors

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 The packaging capacity of adeno-associated virus (AAV) and the potential forwild-type-plusAAV gene therapy vectors

FEBS Letters ◽  
1997 ◽  
Vol 407 (1) ◽  
pp. 78-84 ◽  
Author(s):  
Paul L. Hermonat ◽  
J.Gerald Quirk ◽  
Brian M. Bishop ◽  
Li Han
Keyword(s):  
Gene Therapy ◽  
Adeno Associated Virus ◽  
Gene Therapy Vectors ◽  
Packaging Capacity

scholarly journals Enhancement of Muscle Gene Delivery with Pseudotyped Adeno-Associated Virus Type 5 Correlates with Myoblast Differentiation

2001 ◽  
Vol 75 (16) ◽  
pp. 7662-7671 ◽  
Author(s):  
Dongsheng Duan ◽  
Ziying Yan ◽  
Yongping Yue ◽  
Wei Ding ◽  
John F. Engelhardt
Keyword(s):  
Gene Therapy ◽  
Clinical Trials ◽  
Gene Delivery ◽  
Muscle Cells ◽  
Therapeutic Benefit ◽  
Transduction Efficiency ◽  
Great Promise ◽  
Adeno Associated Virus ◽  
Muscle Gene ◽  
Duchenne's Muscular Dystrophy

ABSTRACT Adeno-associated virus (AAV)-based muscle gene therapy has achieved tremendous success in numerous animal models of human diseases. Recent clinical trials with this vector have also demonstrated great promise. However, to achieve therapeutic benefit in patients, large inocula of virus will likely be necessary to establish the required level of transgene expression. For these reasons, efforts aimed at increasing the efficacy of AAV-mediated gene delivery to muscle have the potential for improving the safety and therapeutic benefit in clinical trials. In the present study, we compared the efficiency of gene delivery to mouse muscle cells for recombinant AAV type 2 (rAAV-2) and rAAV-2cap5 (AAV-2 genomes pseudo-packaged into AAV-5 capsids). Despite similar levels of transduction by these two vectors in undifferentiated myoblasts, pseudotyped rAAV-2cap5 demonstrated dramatically enhanced transduction in differentiated myocytes in vitro (>500-fold) and in skeletal muscle in vivo (>200-fold) compared to rAAV-2. Serotype-specific differences in transduction efficiency did not directly correlate with viral binding to muscle cells but rather appeared to involve endocytic or intracellular barriers to infection. Furthermore, application of this pseudotyped virus in a mouse model of Duchenne's muscular dystrophy also demonstrated significantly improved transduction efficiency. These findings should have a significant impact on improving rAAV-mediated gene therapy in muscle.

scholarly journals Overcoming the Cystic Fibrosis Sputum Barrier to Leading Adeno-associated Virus Gene Therapy Vectors

2014 ◽  
Vol 22 (8) ◽  
pp. 1484-1493 ◽  
Author(s):  
Benjamin S Schuster ◽  
Anthony J Kim ◽  
Joshua C Kays ◽  
Mia M Kanzawa ◽  
William B Guggino ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Therapy ◽  
Adeno Associated Virus ◽  
Virus Gene ◽  
Cystic Fibrosis Sputum ◽  
Gene Therapy Vectors

Targeted Modifications in Adeno-Associated Virus Serotype (AAV)- 8 Capsid Improves Its Hepatic Gene Transfer Efficiency in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2045-2045
Author(s):  
Dwaipayan Sen ◽  
Nishanth Gabriel ◽  
Sathish Kumar Yesupatham ◽  
Rekha Samuel ◽  
Rupali A Gadkari ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Conflicts Of Interest ◽  
Innate Immune ◽  
Hemophilia B ◽  
Transduction Efficiency ◽  
Egfp Expression ◽  
Adeno Associated Virus ◽  
Virus Serotype ◽  
Antibody Levels

Abstract Abstract 2045 Recombinant adeno-associated virus vectors based on serotype (AAV)-8 have shown significant promise for liver directed gene therapy of hemophilia B. However, in a recent clinical trial, two patients who received highest dose (2×1012 vg/kg) of the self-complementary (sc)AAV8 vector developed capsid specific T cells that required glucocorticoid therapy to attenuate this response [Nathwani et al, New Eng J Med, 2011]. Thus, the theme of AAV vector dose dependent immunotoxicity seen with AAV2 vectors earlier seem to re-emerge with AAV8 vectors as well. It is therefore important to develop novel AAV8 vectors that provide enhanced gene expression at significantly less vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal degradation machinery, we modified specific serine/threonine kinase or ubiquitination targets on AAV8 capsid to improve its transduction efficiency. To test this, point mutations at specific serine (S), threonine (T) or lysine (K) residues were generated on AAV8 capsid. scAAV8-EGFP vectors containing the wild-type (WT) and each one of the 5 S/T/K-mutant capsids were evaluated for their liver transduction efficiency at a dose of 5 × 1010 vgs/ animal in C57BL/6 mice in vivo. Two of the AAV8-S>A mutants (S279A and S501A) and a K137R mutant vector, demonstrated significantly higher EGFP expression (3.6 to 12.5 fold) in the liver compared to animals that received WT-AAV8 vectors alone (Figure 1). The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response [interleukin (IL)-6, IL-12, tumor necrosis factor α, Kupffer cells (KC) and innate immune responsive toll like receptors (TLR)-9] with a concomitant 2-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector bio-distribution studies also revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (22 fold), lungs (9.7 fold) and muscle (8.4 fold) tissue when compared to WT-AAV8 vectors. Further on-going studies with the optimal mutant scAAV8 vector expressing human coagulation factor IX in murine models of hemophilia B, will demonstrate the feasibility of the use of these novel vectors for potential gene therapy of hemophilia B. Figure 1: Efficacy of novel AAV8 S>A and K>R vectors (A) EGFP expression in hepatocytes 4 weeks post administration of AAV8 vectors in C57BL/6 mice, (B) Neutralization antibody levels against AAV8 vectors (C) Ubiquitination levels of K137R-AAV8 compared to the WT-AAV8 vector. Figure 1:. Efficacy of novel AAV8 S>A and K>R vectors (A) EGFP expression in hepatocytes 4 weeks post administration of AAV8 vectors in C57BL/6 mice, (B) Neutralization antibody levels against AAV8 vectors (C) Ubiquitination levels of K137R-AAV8 compared to the WT-AAV8 vector. Disclosures: No relevant conflicts of interest to declare.

Immune Response Against Gene Therapy Vectors: Influence of Synovial Fluid on Adeno-Associated Virus Mediated Gene Transfer to Chondrocytes

2004 ◽  
Vol 24 (2) ◽  
pp. 162-169 ◽  
Author(s):  
Virginie Cottard ◽  
Chiara Valvason ◽  
Géraldine Falgarone ◽  
Didier Lutomski ◽  
Marie-Christophe Boissier ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Immune Response ◽  
Gene Transfer ◽  
Synovial Fluid ◽  
Adeno Associated Virus ◽  
Gene Therapy Vectors

scholarly journals The Structure of an AAV5-AAVR Complex at 2.5 Å Resolution: Implications for Cellular Entry and Immune Neutralization of AAV Gene Therapy Vectors

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1326
Author(s):  
Mark A. Silveria ◽  
Edward E. Large ◽  
Grant M. Zane ◽  
Tommi A. White ◽  
Michael S. Chapman
Keyword(s):  
Gene Therapy ◽  
Binding Sites ◽  
Polycystic Kidney ◽  
Adeno Associated Virus ◽  
Gene Therapies ◽  
The Us ◽  
Gene Therapy Vectors ◽  
Cellular Entry ◽  
Resolution Structure

Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology is still not yet fully understood. It has been shown that different serotypes of AAV bind to their cellular receptor, AAVR, in different ways. Previously we have reported a 2.4Å structure of AAV2 bound to AAVR that shows ordered structure for only one of the two AAVR domains with which AAV2 interacts. In this study we present a 2.5Å resolution structure of AAV5 bound to AAVR. AAV5 binds to the first polycystic kidney disease (PKD) domain of AAVR that was not ordered in the AAV2 structure. Interactions of AAV5 with AAVR are analyzed in detail, and the implications for AAV2 binding are explored through molecular modeling. Moreover, we find that binding sites for the antibodies ADK5a, ADK5b, and 3C5 on AAV5 overlap with the binding site of AAVR. These insights provide a structural foundation for development of gene therapy agents to better evade immune neutralization without disrupting cellular entry.

Sign in / Sign up

Export Citation Format

Share Document