Site-Directed Mutagenesis of Surface-Exposed Lysine Residues Leads to Improved Transduction by AAV2, But Not AAV8, Vectors in Murine Hepatocytes <i>In Vivo</i>

Li, Baozheng; Ma, Wenqin; Chen, Ling; Vliet, Kim Van; Huang, Lin; Agbandje‐McKenna, Mavis; Srivastava, Arun; Aslanidi, George

doi:10.1089/hgtb.2015.115

Cited by 29 publications

(34 citation statements)

References 43 publications

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“…administration. These data are consistent with our recently published studies, 62 suggesting that the ubiquitination/proteasome machinery involved in processing the Y-F mutant AAV8 vectors might be different between pancreas and liver, and that the use of lower dose of Y447F+Y733F-AAV8 might also minimize undesired host immune responses to the WT-AAV8 vectors, as has been occasionally observed in human clinical trial. 20,58 Prior whole body distribution studies have observed a discrepancy between the AAV vg copy number and overall gene expression delivered by AAV vectors.…”

Section: Discussionsupporting

confidence: 92%

Efficient Gene Delivery and Expression in Pancreas and Pancreatic Tumors by Capsid-Optimized AAV8 Vectors

Chen

Maeng

Nawab

et al. 2017

Human Gene Therapy Methods

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Despite efforts to use adeno-associated viral (AAV) vector-mediated gene therapy for treatment of pancreatic ductal adenocarcinoma (PDAC), transduction efficiency remains a limiting factor and thus improvement of AAV delivery would significantly facilitate the treatment of this malignancy. Site-directed mutagenesis of specific tyrosine (Y) residues to phenylalanine (F) on the surface of various AAV serotype capsids has been reported as a method for enhancing gene transfer efficiencies. In the present studies, we determine whether Y-to-F mutations could also enhance AAV8 gene transfer in the pancreas to facilitate gene therapy for PDAC. Three different Y-to-F mutant vectors (a single-mutant, Y733F; a double-mutant, Y447F+Y733F; and a triple-mutant, Y275F+Y447F+Y733F) and wild-type AAV8 (WT-AAV8) were administered by intraperitoneal or tail-vein routes to Kras, Kras/Pten, and wild-type mice. The transduction efficiency of these vectors expressing the mCherry reporter gene was evaluated 2 weeks post administration in pancreas or PDAC and correlated with viral genome copy numbers. Our comparative and quantitative analyses of the transduction profiles demonstrated that the Y-to-F double-mutant exhibited the highest mCherry expression in pancreatic tissues (range 45-70%) compared with WT-AAV8 (7%; p < 0.01). We also detected a 7-fold higher level of vector genome copy numbers in normal pancreas following transduction with the double-mutant AAV8 compared with WT-AAV8 (10,285 vs. 1,500 vector copies/μg DNA respectively, p < 0.05). In addition, we observed that intraperitoneal injection of the double-mutant AAV8 led to a 15-fold enhanced transduction efficiency as compared to WT-AAV8 in mouse PDAC, with a corresponding ∼14-fold increase in vector genome copy numbers (26,575 vs. 2,165 copies/μg DNA respectively, p < 0.05). These findings indicate that the Y447+Y733F-AAV8 leads to a significant enhancement of transduction efficiency in both normal and malignant pancreatic tissues, suggesting the potential use of this vector in targeting pancreatic diseases in general, and PDAC in particular.

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Section: Discussionsupporting

confidence: 92%

Efficient Gene Delivery and Expression in Pancreas and Pancreatic Tumors by Capsid-Optimized AAV8 Vectors

Chen

Maeng

Nawab

et al. 2017

Human Gene Therapy Methods

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“…Modification of capsid surface lysine residues also increased transduction efficiency of AAV2 vectors. 75 Tyrosine-modified AAV vectors have been used in clinical trials 76 and efficiently deliver genes to the retina and TM in rodents. Thus, proteasome inhibition or avoidance is one potential way to increase transduction efficiency with viral vectors.…”

Section: Discussionmentioning

confidence: 99%

Proteasome Inhibition Increases the Efficiency of Lentiviral Vector-Mediated Transduction of Trabecular Meshwork

Aktaş

Rao

Slauson

et al. 2018

Invest. Ophthalmol. Vis. Sci.

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PurposeTo determine if proteasome inhibition using MG132 increased the efficiency of FIV vector–mediated transduction in human trabecular meshwork (TM)-1 cells and monkey organ-cultured anterior segments (MOCAS).MethodsTM-1 cells were pretreated for 1 hour with 0.5% dimethyl sulfoxide (DMSO; vehicle control) or 5 to 50 μM MG132 and transduced with FIV.GFP (green fluorescent protein)– or FIV.mCherry-expressing vector at a multiplicity of transduction (MOT) of 20. At 24 hours, cells were fixed and stained with antibodies for GFP, and positive cells were counted, manually or by fluorescence-activated cell sorting (FACS). Cells transduced with FIV.GFP particles alone were used as controls. The effect of 20 μM MG132 treatment on high- and low-dose (2 × 107 and 0.8 × 107 transducing units [TU], respectively) FIV.GFP transduction with or without MG132 was also evaluated in MOCAS using fluorescence microscopy. Vector genome equivalents in cells and tissues were quantified by quantitative (q)PCR on DNA.ResultsIn the MG132 treatment groups, there was a significant dose-dependent increase in the percentage of transduced cells at all concentrations tested. Vector genome equivalents were also increased in TM-1 cells treated with MG132. Increased FIV.GFP expression in the TM was also observed in MOCAS treated with 20 μM MG132 and the high dose of vector. Vector genome equivalents were also significantly increased in the MOCAS tissues. Increased transduction was not seen with the low dose of virus.ConclusionsProteasome inhibition increased the transduction efficiency of FIV particles in TM-1 cells and MOCAS and may be a useful adjunct for delivery of therapeutic genes to the TM by lentiviral vectors.

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“…The elegant simplicity of rAAV vectors has led many researchers to focus on creating new derivatives of this system for gene transfer (100). A naked icosahedral capsid of ~25 nm in diameter can hold ~5 kb of single‐stranded DNA, which seems to be the minimal unit of transfer of genetic material that is likely to result in the transfer of a full‐expression cassette.…”

Section: Future Directionsmentioning

confidence: 99%

The rapidly evolving state of gene therapy

Gruntman

Flotte

2018

FASEB j.

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Gene therapy is emerging as a viable option for clinical therapy of monogenic disorders and other genetically defined diseases, with approved gene therapies available in Europe and newly approved gene therapies in the United States. In the past 10 years, gene therapy has moved from a distant possibility, even in the minds of much of the scientific community, to being widely realized as a valuable therapeutic tool with wide‐ranging potential. The U.S. Food and Drug Administration has recently approved Luxturna (Spark Therapeutics Inc, Philadelphia, PA, USA), a recombinant adeno‐associated virus (rAAV) 2 gene therapy for one type of Leber congenital amaurosis 2 (1, 2). The European Medicines Agency (EMA) has approved 3 recombinant viral vector products: Glybera (UniQure, Amsterdam, The Netherlands), an rAAV vector for lipoprotein lipase deficiency; Strimvelis (Glaxo Smith‐Kline, Brentford, United Kingdom), an ex vivo gammaretrovirus‐based therapy for patients with adenosine deaminase‐deficient severe combined immune deficiency (ADA‐SCID); and Kymriah (Novartis, Basel, Switzerland), an ex vivo lentivirus‐based therapy to engineer autologous chimeric antigen‐receptor T (CAR‐T) cells targeting CD19–positive cells in acute lymphoblastic leukemia. These examples will be followed by the clinical approval of other gene therapy products as this field matures. In this review we provide an overview of the state of gene therapy by discussing where the field stands with respect to the different gene therapy vector platforms and the types of therapies that are available.— Gruntman, A. M., Flotte, T. R. The rapidly evolving state of gene therapy. FASEB J. 32, 1733–1740 (2018). http://www.fasebj.org

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Site-Directed Mutagenesis of Surface-Exposed Lysine Residues Leads to Improved Transduction by AAV2, But Not AAV8, Vectors in Murine Hepatocytes In Vivo

Cited by 29 publications

References 43 publications

Efficient Gene Delivery and Expression in Pancreas and Pancreatic Tumors by Capsid-Optimized AAV8 Vectors

Efficient Gene Delivery and Expression in Pancreas and Pancreatic Tumors by Capsid-Optimized AAV8 Vectors

Proteasome Inhibition Increases the Efficiency of Lentiviral Vector-Mediated Transduction of Trabecular Meshwork

The rapidly evolving state of gene therapy

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