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

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

Journal of Virology ◽

10.1128/jvi.02953-15 ◽

2016 ◽

Vol 90 (16) ◽

pp. 7019-7031 ◽

Cited By ~ 22

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.

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Exosome as a Natural Gene Delivery Vector for Cancer Treatment

Current Cancer Drug Targets ◽

10.2174/1568009620666200924154149 ◽

2020 ◽

Vol 20 (11) ◽

pp. 821-830

Author(s):

Prasad Pofali ◽

Adrita Mondal ◽

Vaishali Londhe

Keyword(s):

Gene Therapy ◽

Gene Delivery ◽

Nucleic Acids ◽

Cancer Treatment ◽

Intestinal Barrier ◽

Gene Carrier ◽

Gene Delivery Vector ◽

Delivery Vector

Background: Current gene therapy vectors such as viral, non-viral, and bacterial vectors, which are used for cancer treatment, but there are certain safety concerns and stability issues of these conventional vectors. Exosomes are the vesicles of size 40-100 nm secreted from multivesicular bodies into the extracellular environment by most of the cell types in-vivo and in-vitro. As a natural nanocarrier, exosomes are immunologically inert, biocompatible, and can cross biological barriers like the blood-brain barrier, intestinal barrier, and placental barrier. Objective: This review focusses on the role of exosome as a carrier to efficiently deliver a gene for cancer treatment and diagnosis. The methods for loading of nucleic acids onto the exosomes, advantages of exosomes as a smart intercellular shuttle for gene delivery and therapeutic applications as a gene delivery vector for siRNA, miRNA and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and also the limitations of exosomes as a gene carrier are all reviewed in this article. Methods: Mostly, electroporation and chemical transfection are used to prepare gene loaded exosomes. Results: Exosome-mediated delivery is highly promising and advantageous in comparison to the current delivery methods for systemic gene therapy. Targeted exosomes, loaded with therapeutic nucleic acids, can efficiently promote the reduction of tumor proliferation without any adverse effects. Conclusion: In the near future, exosomes can become an efficient gene carrier for delivery and a biomarker for the diagnosis and treatment of cancer.

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Abstract MP165: Exosome-mediated Encapsulation Alters AAV Antigenicity and Infectivity: Implications for Gene Delivery in the Heart

Circulation Research ◽

10.1161/res.127.suppl_1.mp165 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Marta Adamiak ◽

Yaxuan Liang ◽

Cherrie Sherman ◽

Shweta Lodha ◽

Erik Kohlbrenner ◽

...

Keyword(s):

Gene Therapy ◽

Gene Delivery ◽

Cardiac Function ◽

Neutralizing Antibodies ◽

Firefly Luciferase ◽

Interferometric Imaging ◽

Human Cardiomyocytes ◽

Clinical Benefits

Gene therapy is a promising approach for the treatment of cardiovascular disease. Current strategies for myocardial gene transfer include the use of adeno-associated virus (AAV) vectors. However, AAVs may not be ideal for gene therapy vectors owing to pre-existing AAV capsid immunity in the human population that may reduce transduction efficacy and hinder preclinical-to-clinical translation. Interestingly, recent studies suggest that exosome-mediated encapsulation may protect viruses from neutralizing antibodies (NAbs) against the capsid and promote viral infectivity. Here, we describe the ability of exosome-enveloped AAVs, i.e. exosomal AAVs (eAAVs), to evade NAbs and serve as a highly efficient gene delivery tool for cardiovascular therapeutics. We have developed a method to purifiy eAAVs from AAV-producing HEK-293T cells, and used electron/confocal microscopy, qPCR, immunoblotting, dynamic light scattering and interferometric imaging measurements to characterize eAAV morphology, contents and mechanism of action. We confirmed eAAVs represent vesicular fractions that exhibit common exosome phenotype, along with the presence of virus particles, and demonstrated that eAAV infectious entry potentially involves trafficking via endocytic compartments. Using flow cytometry, Langendorff perfusion system and bioluminescence imaging, we then evaluated efficiency of heart targeting for eAAV9/eAAV6 and standard AAV9/AAV6 encoding for mCherry or firefly luciferase in human cardiomyocytes in vitro and in mouse model in vivo . Regardless of the presence or absence of NAbs, we showed that eAAVs are more efficient in transduction in the same titer ranges as compared to standard AAVs. To test therapeutic efficacy, we intramyocardially injected eAAV9 or AAV9 vectors encoding for SERCA2a in NAb+ post-myocardial infarction mice and further evaluated cardiac function using echocardiography. Remarkably, eAAV9-SERCA2a outperformed standard AAVs significantly improving cardiac function in the presence of NAbs (%EF 55.14 ± 3.50 compared to 27.31 ± 1.63 at 6 weeks, respectively). In summary, delivery of AAVs protected by carrier exosomes (i.e. eAAVs) may retain the clinical benefits of AAVs while addressing one of its major challenges.

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Adeno-Associated Virus Mediated Gene Delivery: Implications for Scalable in vitro and in vivo Cardiac Optogenetic Models

Frontiers in Physiology ◽

10.3389/fphys.2019.00168 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 16

Author(s):

Christina M. Ambrosi ◽

Gouri Sadananda ◽

Julie L. Han ◽

Emilia Entcheva

Keyword(s):

Gene Delivery ◽

Adeno Associated Virus

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Adeno-Associated Viral Vectors for Gene Transfer and Gene Therapy

Biological Chemistry ◽

10.1515/bc.1999.078 ◽

1999 ◽

Vol 380 (6) ◽

Cited By ~ 63

Author(s):

H. Büeler

Keyword(s):

Gene Expression ◽

Gene Therapy ◽

Gene Transfer ◽

Gene Delivery ◽

Viral Vectors ◽

Delivery Systems ◽

Adeno Associated Virus ◽

Viral Genes ◽

Virus Recombinant

AbstractAdeno-associated virus (AAV) is a defective, non-pathogenic human parvovirus that depends for growth on coinfection with a helper adenovirus or herpes virus. Recombinant adeno-associated viruses (rAAVs) have attracted considerable interest as vectors for gene therapy. In contrast to other gene delivery systems, rAAVs lack all viral genes and show long-term gene expression

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

Journal of Virology ◽

10.1128/jvi.00878-06 ◽

2006 ◽

Vol 80 (19) ◽

pp. 9831-9836 ◽

Cited By ~ 264

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.

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Suppressions of chronic glomerular injuries and TGF-β1 production by HGF in attenuation of murine diabetic nephropathy

AJP Renal Physiology ◽

10.1152/ajprenal.00199.2003 ◽

2004 ◽

Vol 286 (1) ◽

pp. F134-F143 ◽

Cited By ~ 69

Author(s):

Shinya Mizuno ◽

Toshikazu Nakamura

Keyword(s):

Diabetic Nephropathy ◽

Growth Factor ◽

Renal Dysfunction ◽

Transforming Growth Factor ◽

Mesangial Cells ◽

Renal Diseases ◽

Glomerular Mesangial Cells ◽

Diabetic Mice

Diabetic nephropathy is now the leading cause of end-stage renal diseases, and glomerular sclerotic injury is an initial event that provokes renal dysfunction during processes of diabetes-linked kidney disease. Growing evidence shows that transforming growth factor-β1 (TGF-β1) plays a key role in this process, especially in eliciting hypertrophy and matrix overaccumulation. Thus it is important to find a ligand system to antagonize the TGF-β1-mediated pathogenesis under high-glucose conditions. Herein, we provide evidence that hepatocyte growth factor (HGF) targets mesangial cells, suppresses TGF-β1 production, and minimizes glomerular sclerotic changes, using streptozotocin-induced diabetic mice. In our murine model, glomerular sclerogenesis (such as tuft area expansion and collagen deposition) progressed between 6 and 10 wk after the induction of hyperglycemia, during a natural course of diabetic disease. Glomerular HGF expression levels in the diabetic kidney transiently increased but then declined below a basal level, with manifestation of glomerular sclerogenesis. When anti-HGF IgG was injected into mice for 2 wk (i.e., from weeks 4 to 6 after onset of hyperglycemia), these glomerular changes were significantly aggravated. When recombinant HGF was injected into the mice for 4 wk (i.e., between 6 and 10 wk following streptozotocin treatment), the progression of glomerular hypertrophy and sclerosis was almost completely inhibited, even though glucose levels remained unchanged (>500 mg/dl). Even more important, HGF repressed TGF-β1 production in glomerular mesangial cells even under hyperglycemic conditions both in vitro and in vivo. Consequently, not only albuminuria but also tubulointerstitial fibrogenesis were attenuated by HGF. Overall, HGF therapy inhibited the onset of renal dysfunction in the diabetic mice. On the basis of these findings, we wish to emphasize that HGF plays physiological and therapeutic roles in blocking renal fibrogenesis during a course of diabetic nephropathy.

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Identification of an Adeno-Associated Virus Rep Protein Binding Site in the Adenovirus E2a Promoter

Journal of Virology ◽

10.1128/jvi.79.1.28-38.2005 ◽

2005 ◽

Vol 79 (1) ◽

pp. 28-38 ◽

Cited By ~ 10

Author(s):

John M. Casper ◽

Jennifer M. Timpe ◽

John David Dignam ◽

James P. Trempe

Keyword(s):

Gene Expression ◽

Random Sequence ◽

Gene Promoter ◽

Helper Virus ◽

Host Cells ◽

Mobility Shift ◽

Adeno Associated Virus ◽

Rep Protein

ABSTRACT Adeno-associated virus (AAV) and other parvoviruses inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we investigated AAV's interaction with adenovirus (Ad), AAV's most efficient helper virus. Coinfection with Ad and AAV results in an AAV-mediated inhibition of Ad5 gene expression and replication. The AAV replication proteins (Rep) activate and repress gene expression from AAV and heterologous transcription promoters. To investigate the role of Rep proteins in the suppression of Ad propagation, we performed chromatin immunoprecipitation analyses that demonstrated in vivo AAV Rep protein interaction with the Ad E2a gene promoter. In vitro binding of purified AAV Rep68 protein to the Ad E2a promoter was characterized by electrophoretic mobility shift assays (Kd = 200 ± 25 nM). A 38 bp, Rep68-protected region (5′-TAAGAGTCAGCGCGCAGTATTTACTGAAGAGAGCCT-3′) was identified by DNase I footprint analysis. The 38-bp protected region contains the weak E2a TATA box, sequence elements that resemble the Rep binding sites identified by random sequence oligonucleotide selection, and the transcription start site. These results suggest that Rep binding to the E2a promoter contributes to the inhibition of E2a gene expression from the Ad E2a promoter and may affect Ad replication.

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A Chimeric Protein Containing the N Terminus of the Adeno-Associated Virus Rep Protein Recognizes Its Target Site in an In Vivo Assay

Journal of Virology ◽

10.1128/jvi.74.5.2372-2382.2000 ◽

2000 ◽

Vol 74 (5) ◽

pp. 2372-2382 ◽

Cited By ~ 28

Author(s):

Toni Cathomen ◽

Delphine Collete ◽

Matthew D. Weitzman

Keyword(s):

Gene Expression ◽

Mammalian Cells ◽

Target Site ◽

Yeast Genome ◽

Chimeric Proteins ◽

Adeno Associated Virus ◽

Amino Terminal ◽

N Terminus

ABSTRACT The Rep78 and Rep68 proteins of adeno-associated virus (AAV) type 2 are involved in DNA replication, regulation of gene expression, and targeting site-specific integration. They bind to a specific Rep recognition sequence (RRS) found in both the viral inverted terminal repeats and the AAVS1 integration locus on human chromosome 19. Previous in vitro studies implied that an N-terminal segment of Rep is involved in DNA recognition, while additional domains might stabilize binding and mediate multimerization. In order to define the minimal requirements for Rep to recognize its target site in the human genome, we developed one-hybrid assays in which DNA-protein interactions are detected in vivo. Chimeric proteins consisting of the N terminus of Rep fused to different oligomerization motifs and a transcriptional activation domain were analyzed for oligomerization, DNA binding, and activation of reporter gene expression. Expression of reporter genes was driven from RRS motifs cloned upstream of minimal promoters and examined in mammalian cells from transfected plasmids and inSaccharomyces cerevisiae from a reporter cassette integrated into the yeast genome. Our results show for the first time that chimeric proteins containing the amino-terminal 244 residues of Rep are able to target the RRS in vitro and in vivo when incorporated into artificial multimers. These studies suggest that chimeric proteins may be used to harness the unique targeting feature of AAV for gene therapy applications.

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