Arkasubhra Ghosh

Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors

Although adeno-associated virus (AAV)-mediated gene therapy has been hindered by the small viral packaging capacity of the vector, trans-splicing AAV vectors are able to package twice the size of the vector genome. Unfortunately, the efficiency of current trans-splicing vectors is very low. Here we show that rational design of the gene splitting site has a profound influence on trans-splicing vector-mediated gene expression. Using mRNA accumulation as a guide, we generated a set of efficient trans-splicing vectors and achieved widespread expression of the 6-kb ΔH2-R19 mini-dystrophin gene in skeletal muscle of mdx mice, a model for Duchenne muscular dystrophy. The dystrophic phenotype was ameliorated in both adult and aged mice. This demonstrates the use of trans-splicing vectors to efficiently express a large therapeutic structural protein. This strategy should be applicable to other large therapeutic genes or large transcription regulatory elements.

A Single Intravenous Injection of Adeno-associated Virus Serotype-9 Leads to Whole Body Skeletal Muscle Transduction in Dogs

The success of many gene therapy applications hinges on efficient whole body transduction. In the case of muscular dystrophies, a therapeutic vector has to reach every muscle in the body. Recent studies suggest that vectors based on adeno-associated virus (AAV) are capable of body-wide transduction in rodents. However, translating this finding to large animals remains a challenge. Here we explored systemic gene delivery with AAV serotype-9 (AAV-9) in neonatal dogs. Previous attempts to directly deliver AAV to adult canine muscle have yielded minimal transduction due to a strong cellular immune response. However, in neonatal dogs we observed robust skeletal muscle transduction throughout the body after a single intravenous injection. Importantly, systemic transduction was achieved in the absence of pharmacological intervention or immune suppression and it lasted for at least 6 months (the duration of study). We also observed several unique features not predicted by murine studies. In particular, cardiac muscle was barely transduced in dogs. Many muscular dystrophy patients can be identified by neonatal screening. The technology described here may lead to an effective early intervention in these patients.

Viral serotype and the transgene sequence influence overlapping adeno-associated viral (AAV) vector-mediated gene transfer in skeletal muscle

The overlapping approach was developed recently to expand the adeno‐associated viral (AAV) packaging capacity. In this approach, a gene is split into two partially overlapping fragments and separately packaged into an upstream and a downstream vector, respectively. Transgene expression is achieved in co‐infected cells after homologous recombination. Despite the promising proof‐of‐principle results in the lung, the efficiency has been very disappointing in skeletal muscle. Here we examined two potential rate‐limiting factors including AAV serotype and the transgene sequence.

Nanomaterial processing using self-assembly-bottom-up chemical and biological approaches

Nanotechnology is touted as the next logical sequence in technological evolution. This has led to a substantial surge in research activities pertaining to the development and fundamental understanding of processes and assembly at the nanoscale. Both top-down and bottom-up fabrication approaches may be used to realize a range of well-defined nanostructured materials with desirable physical and chemical attributes. Among these, the bottom-up self-assembly process offers the most realistic solution toward the fabrication of next-generation functional materials and devices. Here, we present a comprehensive review on the physical basis behind self-assembly and the processes reported in recent years to direct the assembly of nanoscale functional blocks into hierarchically ordered structures. This paper emphasizes assembly in the synthetic domain as well in the biological domain, underscoring the importance of biomimetic approaches toward novel materials. In particular, two important classes of directed self-assembly, namely, (i) self-assembly among nanoparticle-polymer systems and (ii) external field-guided assembly are highlighted. The spontaneous self-assembling behavior observed in nature that leads to complex, multifunctional, hierarchical structures within biological systems is also discussed in this review. Recent research undertaken to synthesize hierarchically assembled functional materials have underscored the need as well as the benefits harvested in synergistically combining top-down fabrication methods with bottom-up self-assembly.

Efficient Transgene Reconstitution with Hybrid Dual AAV Vectors Carrying the Minimized Bridging Sequences

A hybrid dual-vector system was developed recently as a universal platform to double the packaging capacity of recombinant adeno-associated virus (AAV). In this system, the expression cassette is split into two independent AAV vectors. A highly recombinogenic bridging DNA sequence is engineered in both vectors to mediate target gene-independent homologous recombination between the split vector genomes. In the prototype hybrid vectors, a 0.87-kb DNA fragment from the middle portion of the human placental alkaline phosphatase (AP) gene was used as the bridging sequence. Here we report the development of the minimized bridging sequences. Five independent bridging sequences (0.26 to 0.44 kb) were evaluated in MO59K cells and/or murine skeletal muscle in the context of the AP overlapping vectors and/or the β-galactosidase (LacZ) hybrid vectors. Robust reconstitution comparable to that of the original hybrid vectors was achieved from a 0.26-kb and a 0.27-kb bridging sequence. These newly developed bridging sequences greatly expand the utility of the hybrid dual AAV vector system for delivering larger therapeutic genes/expression cassettes.

Elevated expression of matrix metalloproteinase-9 and inflammatory cytokines in keratoconus patients is inhibited by cyclosporine A.

PURPOSE The present study was designed to understand the role of inflammatory cytokines secreted by corneal epithelial cells in keratoconus (KC) and the response to treatment with cyclosporine A (CyA). METHODS The study involved 129 Indian KC patients clinically graded according to Amsler-Krumeich classification and 20 healthy, nonectatic subjects as controls. Tear levels of matrix metalloproteinase-9 (MMP9), interleukin-6 (IL6), and tumor necrosis factor-α (TNFα) were measured using ELISA kits. Gene expression was measured by qPCR in corneal epithelial cells obtained by debridement from subjects undergoing ocular surface surgeries. In addition, epithelial cells were stimulated with TNFα and treated with CyA to study its role on MMP9 expression. Finally, 20 KC patients (27 eyes) with inflammatory symptoms were treated with topical CyA application. RESULTS We observed that MMP9, TNFα, and IL6 levels were strongly upregulated at the mRNA level in KC patient epithelia. Similarly, tears collected from KC patients exhibited high levels of MMP9 and IL6 protein. Cyclosporine A treatment significantly reduced the mRNA expression levels of IL6 and TNFα in both short- and long-term treatments; however, it reduced MMP9 levels only in long-term treatment in cultured corneal epithelial cells. Subsequent treatment of KC patients with CyA for approximately 6 months reduced tear MMP9 levels and led to local reduction in corneal curvatures as determined by corneal topography maps. CONCLUSIONS The data indicate that corneal epithelium contributes to elevated MMP9 and inflammatory cytokine expression in tears of KC patients. Cyclosporine A treatment reduced MMP9 and inflammatory cytokine levels in an in vitro inflammation model system. In KC patients, CyA treatment reduced MMP9 levels measured in tears with concomitant arrest of disease progression. Therefore, CyA might be a novel treatment strategy in KC patients but requires additional evaluation in larger cohorts. (ClinicalTrials.gov number, NCT01746823.).

AAV serotype influences gene transfer in corneal stroma in vivo.

This study evaluated the cellular tropism and relative transduction efficiency of three AAV serotypes, AAV6, AAV8 and AAV9, for corneal gene delivery using mouse cornea in vivo and donor human cornea ex vivo. The AAV6, AAV8 and AAV9 serotypes having AAV2 plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK 293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Viral vectors (10(9) vg/microl) were topically applied onto mouse cornea in vivo and human cornea ex vivo after removing the epithelium. Human corneas were processed for transgene delivery at day 5 after viral vector application. Mouse corneas were harvested at 4, 14 and 30 days after vector application for AP staining. Transduction efficiency was calculated by quantifying pixels of AP-stained area using Image J software and also confirmed by functional AP enzyme activity in the corneal lysates. Cellular toxicity of the three AAV serotypes was tested with TUNEL assay. Inflammatory response was detected by immunostaining for CD11b and F4/80. All three AAV serotypes successfully transduced mouse and human corneas. The order of transduction efficiency was AAV9 > AAV8 > AAV6. The transduction efficiency of AAV9 was 1.1-1.4 fold higher (p > 0.05) as compared to AAV8 and 3.5-5.5 fold higher (p < 0.01) as compared to AAV6. The level of transgene expression for all the three serotypes was greater at 14 days compared to 4 days and this high level of transgene expression was maintained up to the tested time point of 30 days. Corneas exposed to any of the three AAV serotypes did not show significant TUNEL positive cells or any inflammatory response as tested by CD11b or F4/80 staining suggesting that tested AAV serotypes do not induce cell death or inflammation and are safe for corneal gene therapy.

Systemic Trans-Splicing Adeno-Associated Viral Delivery Efficiently Transduces the Heart of Adult mdx Mouse, a Model for Duchenne Muscular Dystrophy

Trans-splicing adeno-associated viral (tsAAV) vectors hold great promise for delivering large therapeutic genes. One potential application is in the treatment of Duchenne muscular dystrophy (DMD). In this case, it is necessary to transduce whole body muscle. We demonstrated body-wide AAV-9 tsAAV transduction in normal neonatal mice. However, it was not clear whether such an approach would work in diseased mice. In this study we delivered the AAV-9 alkaline phosphatase (AP) tsAAV vector (3 x 10(12) vector genome particles per vector per mouse, tail vein injection) to 2-month-old mdx mice, the most widely used DMD model. Four months later, we observed widespread AP expression in the heart. It reached the same level as we have seen in normal neonatal puppy. Interestingly, myocardial transduction correlated with beta-myosin heavy chain expression but not with LamR, the putative AAV-9 receptor. AP expression was also detected in various skeletal muscles but at levels much lower than in normal newborn mice. Despite the existing inflammatory milieu, we did not see any appreciable increase in CD4(+) and CD8(+) T cells and macrophages in striated muscles after systemic tsAAV infection. In summary, our results have paved the way for tsAAV-mediated gene therapy for Duchenne cardiomyopathy.

Transduction efficiency of AAV 2/6, 2/8 and 2/9 vectors for delivering genes in human corneal fibroblasts

In the present study, cellular tropism and relative transduction efficiency of AAV2/6, AAV2/8 and AAV2/9 vectors have been tested for the cornea using primary cultures of human corneal fibroblasts. The AAV6, AAV8 and AAV9 serotypes having AAV2 ITR plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Primary cultures of human corneal fibroblasts were exposed to AAV infectious particles at two different doses (1 x 10(5) and 2 x 10(5) MOI). Cytochemistry and enzyme assays were used to measure delivered transgene expression in samples collected at 4 and 30 h after AAV infection by counting AP-stained cells or quantifying AP enzyme activity. Cellular toxicity of AAVs was evaluated with TUNEL and trypan blue assays. All three AAV serotypes transduced human corneal fibroblasts. The order of transduction efficiency was AAV2/6>>>AAV2/9>AAV2/8. The transduction efficiency of AAV2/6 was 30-50-fold higher (p < 0.001) for the human corneal fibroblasts compared to the AAV2/8 or AAV2/9 at two tested doses. The level of transgene expression at 4h was considerably low compared to 30 h suggesting that the transgene delivery did not reach its peak at 4h. Cultures exposed to any of the three AAV serotypes showed more than 97% cellular viability and less than 5 TUNEL positive cells suggesting that tested AAV serotypes do not induce significant cell death and are safe for corneal gene therapy.

Biomechanics of the Cornea Evaluated by Spectral Analysis of Waveforms from Ocular Response Analyzer and Corvis-ST

Purpose In this study, spectral analysis of the deformation signal from Corvis-ST (CoST) and reflected light intensity from ocular response analyzer (ORA) was performed to evaluate biomechanical concordance with each other. Methods The study was non-interventional, observational, cross-sectional and involved 188 eyes from 94 normal subjects. Three measurements were made on each eye with ORA and CoST each and then averaged for each device. The deformation signal from CoST and reflected light intensity (applanation) signal from ORA was compiled for all the eyes. The ORA signal was inverted about a line joining the two applanation peaks. All the signals were analyzed with Fourier series. The area under the signal curves (AUC), root mean square (RMS) of all the harmonics, lower order (LO included 1st and 2nd order harmonic), higher order (HO up to 6th harmonic), CoST deformation amplitude (DA), corneal hysteresis (CH) and corneal resistance factor (CRF) were analyzed. Results The device variables and those calculated by Fourier transform were statistically significantly different between CoST and ORA. These variables also differed between the eyes of the same subject. There was also statistically significant influence of eyes (left vs. right) on the differences in a sub-set of RMS variables only. CH and CRF differed statistically significantly between the eyes of subject (p<0.001) but not DA (p = 0.65). Conclusions CoST was statistically significantly different from ORA. CoST may be useful in delineating true biomechanical differences between the eyes of a subject as it reports deformation.