Dwaipayan Sen

Improving clinical efficacy of adeno associated vectors by rational capsid bioengineering

Adeno associated vectors (AAV) have shown considerable promise to treat various genetic disorders in both preclinical and clinical settings mainly because of its safety profile. However, efficient use of AAV to deliver genes in immune-competent sites like muscles and liver requires very high doses which are associated with concomitant cellular immune response against the viral capsids leading to destruction of the transduced cells. Coupled with that, there are enough evidences that at high doses, AAV particles are subjected to increased cellular phosphorylation/uniquitination leading to proteasome mediated degradation and loss of the viral particles. The presence of preexisting immunity against AAV further adds on to the problem which is acting as a major roadblock to efficiently use it as a gene therapy vector in the clinics. To overcome this, rational bioengineering of AAV capsid becomes a prime tool by which specific amino acid residue(s) can be suitably modified/replaced by compatible residue(s) to create vectors having lower host immune response and higher intracellular trafficking rate. This article reviews the various aspects of rationally designing AAV capsids like by site-directed mutagenesis, directed evolution and combinatorial libraries which can create vectors having not only immune evasive property but also enhanced gene expression and transduction capability. One or more combinations of these strategies have strong potential to create novel vectors which will have suitable clinical efficiency even at a low dose.

MicroRNAs in Parkinson’s disease

Parkinson’s disease is the second most common neurodegenerative disease commonly affecting the older population. Loss of dopaminergic neurons in the substantia nigra of brain leads to impairment of motor activities as well as cognitive defects. There are many underlying causes to this disease, both genetic and epigenetic, which are yet to be fully explored. Non-coding RNAs are significant part of our genome and are involved in various cellular processes. MicroRNAs, which are small non-coding RNAs having 20–22 nucleotides, are involved in many underlying mechanisms of pathogenesis of several neurodegenerative diseases including Parkinson’s. This review focuses on the role played by microRNAs in regulating various genes responsible for the onset and pathogenesis of Parkinson’s disease and various literature evidences pointing at the usefulness of targeting specific microRNAs as a potential alternate therapeutic strategy for successful impairment of the disease progression. This review also discusses about various biofluid-based microRNA markers which may be potentially utilized for diagnostic purposes.

Promise of adeno-associated virus as a gene therapy vector for cardiovascular diseases

Cardiovascular diseases pose a unique threat to global mortality because it presents as one of the most diverse conglomerations of pathophysiological conditions that can create significant casualty even without straying into its collateral damage. This puts them right beside obesity and cancer in terms of severity. Their pervasive nature and high prevalence prompted biologists to seek newer prophylactic avenues of addressing this global hazard, among which adeno-associated virus (AAV) gene therapy rose to significant prominence. By virtue of its unrivaled clinical safety quotient, AAVs have been used to rectify various subtypes of cardiovascular ailments, beginning from commonly occurring heart failure to vascular diseases. The review focuses on the history of AAV-mediated gene therapy and contemporary breakthroughs in terms of novel innovations in vector engineering to reduce detargeting, immune response, untimely expression, and so on. We have also focused on the molecular world of cardiomyocytes and endothelial cells but interpreted the therapies in a broader context of cardiovascular pathology. The advances made in each mode of intervention as well as the ones that are beyond the scope of AAV gene therapy or has not been approached through AAV gene therapy as of now have been provided in detail to illustrate the bigger picture of where we stand to combat cardiovascular diseases most efficiently.

Can crosstalk between DOR and PARP reduce oxidative stress mediated neurodegeneration

&NA; The progressive loss of structure and function of neurons leads to neurodegenerative processes which become the causative reason for various neurodegenerative diseases such as Parkinsons disease (PD), Alzheimers disease (AD) etc. These diseases are multifactorial in nature but they have been seen to possess similar causative agents to a certain extent. Oxidative Stress (OS) has been identified as a major stressor and a mediator in most of these diseases. OS not only leads to the generation of free radical species but if persistent, can possibly lead to lipid peroxidation, protein damage, DNA damage, and cell death. Anti‐oxidants are endogenously present in our body to tackle oxygen metabolites but their levels reduce greatly under continuous OS conditions. In such a case, dietary supplements to replenish the anti‐oxidant levels in our body is a good way of treatment but it is very slow and may not be as effective in chronic stress conditions. Thus, there is a need for more effective mechanisms to attenuate OS. Two such mechanisms which can be considered are the activation of Delta opioid receptor (DOR) and Inhibition of Poly (ADP‐ribose)‐polymerase1 (PARP1), which have been suggested to protect neurons and increase neuronal cell survivability in both in‐vitro and in‐vivo disease models. Various signaling pathways have been highlighted to probably play a significant role in attenuating OS by the activation of DOR. It would be an interesting topic of investigation to see if one of the probable mechanisms by which DOR attenuates OS could be by modulation of PARP through a cascade of intracellular signaling reactions. HighlightsOxygen metabolism creates oxidant species which leads to oxidative stress.Endogenous and exogenous antioxidants help in attenuating oxidative stress.DOR activation leads to neuroprotection.PARP inhibition increases cell survivability and improves mitochondrial functioning.A DOR mediated mechanism could modulate PARP activity to attenuate oxidative stress.

The delta opioid peptide D-Alanine 2, Leucine 5 Enkephaline (DADLE)-induces neuroprotection through cross-talk between the UPR and pro-survival MAPK-NGF-Bcl2 signaling pathways via modulation of several micro-RNAs in SH-SY5Y cells subjected to ER stress: DADLE mediated neuroprotection in SH-SY5Y cells under ER stress

Parkinsons disease (PD) is the second most progressive neurodegenerative disease characterized by the loss of dopaminergic neurons and accumulation of misfolded proteins in endoplasmic reticulum (ER) leading to activation of the unfolded protein response (UPR). In the present study, we aimed to determine the potential survival effect of the delta opioid neuro‐peptide D‐Alanine 2, Leucine 5 Enkephaline (DADLE), and its mechanism in dopaminergic SH‐SY5Y cells which were subjected to ER stress. In this cellular model of PD, enhanced cell survivability was observed on DADLE treatment (but not with μ and κ opioid agonists) along with concomitant down regulation of the UPR stress sensors and protein aggregates. The study found increased phosphorylation of MEK‐1, which leads to activation of MAP kinase as well as enhanced expression of the pro‐survival gene nerve growth factor and anti‐apoptotic marker Bcl2. DADLE treatment could also significantly inhibit expression of the pro‐apoptotic marker BIM. Next‐generation sequence analysis revealed 93 micro (mi) RNAs to be differentially regulated following DADLE treatment in cells subjected to ER stress. Pathway prediction and previously published reports revealed that out of these 93 miRNAs, 34 can play a role in promoting cell survival. Specific modulation of two such miRNAs, namely miR‐30c‐2‐3p and miR‐200c, could partially reverse the positive survival effect induced by DADLE. Apart from the known miRNAs, various novel miRNAs were also observed following DADLE treatment which could also play a role in enhancing the survival of SH‐SY5Y cells under ER stress.

DADLE enhances viability and anti-inflammatory effect of human MSCs subjected to ‘serum free’ apoptotic condition in part via the DOR/PI3K/AKT pathway

Aim: Nutritional deprivation and inflammation‐rich zones are the major causative reasons for poor survivability of transplanted mesenchymal stem cells (MSCs). Therefore in the present study, we demonstrated the cytoprotective and anti‐inflammatory effects of activated delta (&dgr;)‐opioid receptor (DOR) with synthetic peptide [D‐Ala2, D‐Leu5]‐enkephalin (DADLE) treatment on human MSCs cultured in serum‐starved condition. Main methods: Cell viability was measured using MTT and Annexin V/PI assays. Expressions of pro‐apoptotic (Bcl2) and anti‐apoptotic genes (Bax/Bad), levels of activated p44/42 MAPK, Akt, PI3‐kinase‐p110&ggr; and cleaved caspase‐3 were determined by qPCR and western blot. Levels of secreted cytokines were measured by ELISA. Key findings: In comparison to the control, DADLE significantly increased cell survivability under serum deprived condition as confirmed by MTT (71% vs 45%) and Annexin V/PI assays (25.9% vs 3.7%). Significant up‐regulation of pro‐apoptotic Bcl2 (˜ 2.1 folds), down‐regulations of anti‐apoptotic Bax/Bad (˜ 2.6/2.7 folds) as well as of cleaved caspase‐3, increased expression of PI3kinase subunit p110&ggr; and activation of Akt (Ser473) were observed following DADLE treatment in cells under ‘serum deprivation’ stress. In addition, DADLE treated hMSCs secreted increased levels of anti‐inflammatory cytokines (IL10/IL4/TGF‐&bgr;) under serum deprived condition. LPS stimulated macrophages showed abated release of pro‐inflammatory cytokines (IL1/TNF&agr;/IL6) when grown in hMSC conditioned ‘serum deprived’ media treated with DADLE. Both the cytoprotective and anti‐inflammatory effects of DADLE were inhibited by the DOR specific antagonist naltrindole. Significance: The DOR signaling pathway improved cell viability and enhanced anti‐inflammatory effect of hMSCs subjected to ‘serum deprivation’ stress that could have potential therapeutic benefits in reparative medicine.

Stent-mediated gene and drug delivery for cardiovascular disease and cancer: A brief insight

This review concisely recapitulates the different existing modes of stent‐mediated gene/drug delivery, their considerable advancement in clinical trials and a rationale for other merging new technologies such as nanotechnology and microRNA‐based therapeutics, in addition to addressing the limitations in each of these perpetual stent platforms. Over the past decade, stent‐mediated gene/drug delivery has materialized as a hopeful alternative for cardiovascular disease and cancer in contrast to routine conventional treatment modalities. Regardless of the phenomenal recent developments achieved by coronary interventions and cancer therapies that employ gene and drug‐eluting stents, practical hurdles still remain a challenge. The present review highlights the limitations that each of the existing stent‐based gene/drug delivery system encompasses and therefore provides a vision for the future with respect to discovering an ideal stent therapeutic platform that would circumvent all the practical hurdles witnessed with the existing technology. Further study of the improvisation of next‐generation drug‐eluting stents has helped to overcome the issue of restenosis to some extent. However, current stent formulations fall short of the anticipated clinically meaningful outcomes and there is an explicit need for more randomized trials aiming to further evaluate stent platforms in favour of enhanced safety and clinical value. Gene‐eluting stents may hold promise in contributing new ideas for stent‐based prevention of in‐stent restenosis through genetic interventions by capitalizing on a wide variety of molecular targets. Therefore, the central consideration directs us toward finding an ideal stent therapeutic platform that would tackle all of the gaps in the existing technology.

Mesenchymal Stem Cells as a Source of Dopaminergic Neurons: A Potential Cell Based Therapy for Parkinson's Disease

Cell repair/replacing strategies for neurodegenerative diseases such as Parkinsons disease depend on well-characterized dopaminergic neuronal candidates that are healthy and show promising effect on the rejuvenation of degenerated area of the brain. Therefore, it is imperative to develop innovative therapeutic strategies that replace damaged neurons with new/functional dopaminergic neurons. Although several research groups have reported the generation of neural precursors/neurons from human/ mouse embryonic stem cells and mesenchymal stem cells, the latter is considered to be an attractive therapeutic candidate because of its high capacity for self-renewable, no adverse effect to allogeneic versus autologous transplants, high ethical acceptance and no teratoma formation. Therefore, mesenchymal stem cells can be considered as an ideal source for replacing lost cells in degenerative diseases like Parkinsons. Hence, the use of these cells in the differentiation of dopaminergic neurons becomes significant and thrives as a therapeutic approach to treat Parkinsons disease. Here we highlight the basic biology of mesenchymal stem cells, their differentiation potential into dopaminergic neurons and potential use in the clinics.

The Delta Opioid Peptide DADLE Represses Hypoxia-Reperfusion Mimicked Stress Mediated Apoptotic Cell Death in Human Mesenchymal Stem Cells in Part by Downregulating the Unfolded Protein Response and ROS along with Enhanced Anti-Inflammatory Effect

Hypoxia-reperfusion (H/R) emblems a plethora of pathological conditions which is potent in contributing to the adversities encountered by human mesenchymal stem cells (hMSCs) in post-transplant microenvironment, resulting in transplant failure. D-Alanine 2, Leucine 5 Enkephaline (DADLE)-mediated delta opioid receptor (DOR) activation is well-known for its recuperative properties in different cell types like neuronal and cardiomyocytes. In the current study its effectiveness in assuaging hMSC mortality under H/R-like insult has been delineated. The CoCl2 mimicked H/R conditions in vitro was investigated upon DOR activation, mediated via DADLE. hMSCs loss of viability, reactive oxygen species (ROS) production, inflammatory responses and disconcerted unfolded protein response (UPR) were assessed using AnnexinV/PI flow cytometry, fluorescence imaging, mitochondrial complex 1 assay, quantitative PCR, immunoblot analysis and ELISA. H/R like stress induced apoptosis of hMSCs was significantly mitigated by DADLE via modulation of the apoptotic regulators (Bcl-2/Bax) along with significant curtailment of ROS and mitochondrial complex 1 activity. DADLE concomitantly repressed the misfolded protein aggregation, alongside the major UPR sensors: PERK/BiP/IRE-1α /ATF-6, evoked due to the H/R mimicked endoplasmic reticulum stress. Undermined phosphorylation of the Akt signalling pathway was observed, which concerted its effect onto regulating both the pro and anti-inflammatory cytokines, actuated as a response to the H/R-like insult. The effects of DADLE were subdued by naltrindole (specific DOR antagonist) reaffirming the involvement of DOR in the process. Taken together these results promulgate the role of DADLE-induced DOR activation on improved hMSC survival, which signifies the plausible implications of DOR-activation in cell-transplantation therapies and tissue engineering aspect.

DOR agonist (SNC-80) exhibits anti-parkinsonian effect via downregulating UPR/oxidative stress signals and inflammatory response in vivo

The pathophysiology of Parkinsons disease exhibit imperative roles in unfolded protein response stress-induced oxidative stress and inflammation in general. Although, delta opioid receptor (DOR), has been found to represent anti-parkinsonian effect at behavioral level, its underlying mechanism remains elusive till date. In the present study the role of DOR agonist, SNC-80 and the consorted molecular mechanisms, which translates to behavioral recuperation, has been delineated. In order to mimic PD, mice were intra-peritoneally injected with MPTP, following exposure to SNC-80 and L-DOPA to elucidate amelioration of the MPTP-induced behavioral impairments. The results obtained suggest that the severity of the compromised motor functions up-regulated the UPR stress sensors: IRE-1α/Bip/CHOP, oxidative stress along with the pro-inflammatory cytokines: IL1β/IFNγ/TNFα and IL-6. These inimical factors combined, aids the persistence of the disease in MPTP intoxicated mice. Supplementation with SNC-80 significantly improved motor functions via down-regulation of the UPR stress sensors and inflammatory cytokines. Additionally, SNC-80 could upregulate Nrf-2 and Heme oxygenase-1 (HO-1) protein expression indicating their involvement in SNC-80s potential anti-oxidant function. There was also a significant reduction in protein carbonyl content indicating the positive role of SNC-80 in dampening MPTP induced oxidative stress. Concomitantly, L-DOPA also demonstrated an enhanced effect towards improvement of motor functions but did not suppress the UPR and inflammatory responses caused due to MPTP intoxication. Hence, these results suggest that SNC-80 could hold a pivotal role in replenishing motor functions essentially via regulating UPR and inflammation.