Current
treatment approaches for hepatocellular carcinoma (HCC)
have a narrow therapeutic index and alternate modes of treatment are
thus required. We have utilized a gene delivery vector containing
inducible caspase 9 (iCasp9) gene, which is a synthetic analogue based
on the mammalian caspase 9 and fused to a human FK506 binding protein
that allows its conditional dimerization to a synthetic, small molecule
[chemical inducer of dimerization, AP20187] and results in target
cell apoptosis. In our studies, we have tested these synthetic vectors
based on an adeno-associated virus platform for their potential anti-tumorigenic
effect in human HCC cells in vitro and in a HCC tumor
model developed in nude mice. Our data demonstrates
that the iCasp9-AP20187 bioconjugate is able to trigger terminal effectors
of cellular apoptosis and presents a viable approach for the potential
treatment of HCC.
Recombinant
adeno-associated virus (AAV)-based gene therapy has
been promising, but several host-related transduction or immune challenges
remain. For this mode of therapy to be widely applicable, it is crucial
to develop high transduction and permeating vectors that infect the
target at significantly low doses. Because glycosylation of capsid
proteins is known to be rate limiting in the life cycle of many viruses,
we reasoned that perturbation of glycosylation sites in AAV2 capsid
will enhance gene delivery. In our first set experiments, pharmacological
modulation of the glycosylation status in host cells, modestly decreased
(1-fold) AAV2 packaging efficacy while it improved their gene expression
(∼74%) in vitro. We then generated 24 mutant AAV2 vectors modified
to potentially create or disrupt a glycosylation site in its capsid.
Three of them demonstrated a 1.3–2.5-fold increase in transgene
expression in multiple cell lines (HeLa, Huh7, and ARPE-19). Hepatic
gene transfer of these vectors in hemophilia B mice, resulted in a
2-fold increase in human coagulation factor (F)IX levels, while its
T/B-cell immunogenic response was unaltered. Subsequently, intravitreal
gene transfer of glycosylation site-modified vectors in C57BL6/J mice
demonstrated an increase in green fluorescence protein expression
(∼2- to 4-fold) and enhanced permeation across retina. Subretinal
administration of these modified vectors containing RPE65 gene further
rescued the photoreceptor response in a murine model of Leber congenital
amarousis. Our studies highlight the translational potential of glycosylation
site-modified AAV2 vectors for hepatic and ocular gene therapy applications.
During recombinant Adeno-associated virus (AAV) production, a proportionately large amount of vectors is released in the culture supernatant, which is often discarded. It has been shown that these vectors often associate with vesiculated structures, such as exosomes. Exosome-associated AAV (vexosomes) represent an additional gene-delivery platform. The efficiency of such vexosomes in suicide gene therapy is unexplored. In the present study, we have generated AAV serotype 6 vexosomes containing an inducible caspase 9 (iCasp9) suicide gene by a differential ultracentrifugation-based protocol. We further tested the cytotoxic potential of these vexosomes in a human hepatocellular carcinoma (HCC) model in vitro and in vivo. The AAV6-iCasp9 containing vexosomes, when primed with a pro-drug (AP20187), demonstrated a significant loss in cell viability (57% ± 8% versus 100% ± 4.8%, p < 0.001) in comparison to mock-treated Huh7 cells. An intratumoral administration of AAV6-iCasp9 vexosomes and AP20187 in a murine xenograft model revealed a 2.3-fold increase in tumor regression in comparison to untreated animals. These findings were further corroborated by histological analysis and apoptosis assays. In conclusion, our data demonstrate the therapeutic potential of AAV6 vexosomes in a xenotransplantation model of HCC. Furthermore, the simplicity in production and isolation of vexosomes should further facilitate its application in other malignancies.
Background: Diabetes mellitus (DM) is widely spread in South Asian (ASEAN) and Indian sub-continent. The increasing healthcare costs of DM can be prevented in the developing world by improved public healthcare interventions. Modifiable risk factors of DM like sedentary lifestyle, obesity, and stressful conditions are associated with its progression; however, the epidemiological data collected by Public Institutions are limited. Summary: A review of published literature describing geographic distribution of DM and associated dementia in South Asian region, particularly India, was conducted with the purpose of assessing the feasibility and challenges associated with the Yoga-based risk reduction. PubMed and Google Scholar databases were searched for DM and dementia-related articles by using a combination of keywords: Diabetes, Diabetes related Dementia Southeast Asia, Pre-diabetes, Yoga, lifestyle modification, Dementia and Exercise. The epidemiological data generated from these diseases have not prompted to any major public health policies. Yoga can be a cost-effective intervention for the prevention of Type 2 DM (T2DM) and its associated cognitive decline when detected early. If nationwide intervention of Yoga is brought about by the state, its integration in health care will become more meaningful and acceptable. Key Message: Studies suggest that Yoga and change in lifestyle can modify the health risks associated with T2DM and associated dementia if it is mainstreamed with the public health initiative of Ayushman Bharat scheme.
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