Manika Vij

Integration Host Factor of Mycobacterium tuberculosis, mIHF, Compacts DNA by a Bending Mechanism

The bacterial chromosomal DNA is folded into a compact structure called as ‘nucleoid’ so that the bacterial genome can be accommodated inside the cell. The shape and size of the nucleoid are determined by several factors including DNA supercoiling, macromolecular crowding and nucleoid associated proteins (NAPs). NAPs bind to different sites of the genome in sequence specific or non-sequence specific manner and play an important role in DNA compaction as well as regulation. Until recently, few NAPs have been discovered in mycobacteria owing to poor sequence similarities with other histone-like proteins of eubacteria. Several putative NAPs have now been identified in Mycobacteria on the basis of enriched basic residues or histone-like “PAKK” motifs. Here, we investigate mycobacterial Integration Host Factor (mIHF) for its architectural roles as a NAP using atomic force microscopy and DNA compaction experiments. We demonstrate that mIHF binds DNA in a non-sequence specific manner and compacts it by a DNA bending mechanism. AFM experiments also indicate a dual architectural role for mIHF in DNA compaction as well as relaxation. These results suggest a convergent evolution in the mechanism of E. coli and mycobacterial IHF in DNA compaction.

Non-invasive topical delivery of plasmid DNA to the skin using a peptide carrier.

Topical delivery to skin is an essential step in non-invasive application of nucleic acid therapeutics for cutaneous disorders. The barrier posed by different layers of the skin - stratum corneum on top followed by the viable epidermis below - makes it extremely challenging for large hydrophilic molecules like nucleic acids to efficiently enter the uncompromised skin. We report an amphipathic peptide Mgpe9 (CRRLRHLRHHYRRRWHRFRC) that can penetrate the uncompromised skin, enter skin cells and deliver plasmid DNA efficiently as nanocomplexes in vitro and in vivo without any additional physical or chemical interventions prevalent currently. We observe efficient gene expression up to the highly proliferating basal layer of the skin without observable adverse reactions or toxic effects after delivery of reporter plasmids. The entry mechanism of nanocomplexes possibly involves reversible modulation of junction proteins accompanied by transient changes in skin structure. This peptide holds potential to be used as an efficient transporter of therapeutic nucleic acids to the skin.

Systemic delivery of the tumor necrosis factor gene to tumors by a novel dual DNA-nanocomplex in a nanoparticle system

Many cancers fail to respond to immunotherapy as a result of immune suppression by the tumor microenvironment. The exogenous expression of immune cytokines to reprogram the tumor microenvironment represents an approach to circumvent this suppression. The present studies describe the development of a novel dual nanoparticle (DNP) system for driving DNA expression vectors encoding inflammatory cytokines in tumor cells. The DNP system consists of a DNA expression vector-cationic peptide nanocomplex (NC) surrounded by a diblock polymeric NP. Tumor necrosis factor alpha (TNF) was selected as the prototype cytokine for this system, based on its pleotropic inflammatory and anti-cancer activities. Our results demonstrate that the DNP system is highly effective in driving expression of TNF in tumor cells. We also demonstrate that the DNPs are effective in inducing apoptosis and anti-tumor activity. These findings support a novel immunotherapeutic approach for the intratumoral delivery of DNA vectors that express inflammatory cytokines.

Bioinspired Functionalized Melanin Nanovariants with a Range of Properties Provide Effective Color Matched Photoprotection in Skin.

Melanin and related polydopamine hold great promise; however, restricted fine-tunabilility limits their usefulness in biocompatible applications. In the present study, by taking a biomimetic approach, we synthesize peptide-derived melanin with a range of physicochemical properties. Characterization of these melanin polymers indicates that they exist as nanorange materials with distinct size distribution, shapes, and surface charges. These variants demonstrate similar absorption spectra but have different optical properties that correlate with particle size. Our approach enables incorporation of chemical groups to create functionalized polyvalent organic nanomaterials and enables customization of melanin. Further, we establish that these synthetic variants are efficiently taken up by the skin keratinocytes, display appreciable photoprotection with minimal cytotoxicity, and thereby function as effective color matched photoprotective agents. In effect we demonstrate that an array of functionalized melanins with distinct properties could be synthesized using bioinspired green chemistry, and these are of immense utility in generating customized melanin/polydopamine like materials.

Redox-Dependent Condensation Of Mycobacterial Genome By WhiB4

Conventionally, oxidative stress response in bacteria is mediated through coordination between the regulators of oxidant-remediation systems (e.g. OxyR, SoxR) and nucleoid condensation (e.g. Dps, Fis). However, Mycobacterium tuberculosis (Mtb) lacks these mechanisms. Therefore, how Mtb organizes genome architecture and regulates gene expression to counterbalance oxidative imbalance during infection is not known. Using systems biology and imaging techniques, we report that an intracellular redox-sensor, WhiB4, dynamically regulates genome condensation and multiple oxidative stress response networks in Mtb. Notably, a low degree of oxidative stress induced marginal genome condensation, while heightened oxidative stress triggered mycobacterial death through nucleoid hyper-condensation. Deletion of WhiB4 alleviated, whereas over-expression aggravated the negative impact of DNA condensation on oxidative stress survival of Mtb. Further, our results suggest that WhiB4 mediates both architectural and regulatory roles by controlling auto-expression, homo-interaction, and hetero-interaction with sigma factors, SigE and SigA, in response to changes in intramycobacterial redox potential. Over-expression of WhiB4 in Mtb disrupts redox homeostasis, damages genome integrity, and synergizes with host-generated radicals to exert efficient killing inside macrophages and mice. Expression of SigE counteracted the deleterious influence of WhiB4 over-expression on nucleoid condensation and survival, indicating that WhiB4-SigE constitutes a system that calibrates oxidative stress response in Mtb. We infer that WhiB4 is a novel redox-dependent architectural protein that structurally couples response to oxidative stress with changes in genome organization and transcription in Mtb. This previously unidentified dependence of Mtb on WhiB4 and nucleoid condensation to modulate oxidative stress response expands our understanding of bacterial pathogenicity. Author Summary In pathogenic mycobacteria, understanding of the concerted rearrangements of gene activities during various stages of infection is a fundamental problem. To persist, Mtb needs to adapt in response to an array of successive environmental challenges encountered during infection. Most of the hostile conditions within host, including acidic and oxidative stress, are known to induce changes in DNA topology in other bacterial systems. Variations in nucleoid condensation in response to changing environmental conditions may serve as a signal triggering the virulence program during the infection process. We deciphered a redox-based mechanistic device, WhiB4, coordinating the chromosomal structure with selective expression of the adaptive traits in response to oxidative stress. Using a holistic approach exploring the inherent relationships between the physicochemical properties of the DNA, cytoplasmic redox potential, and regulation of virulence factors network mediating adaptive potential in Mtb, we uncovered a fundamental basis of oxidative stress tolerance and mycobacterial persistence during infection.

Efficient Cellular Entry of (r-x-r)-Type Carbamate–Plasmid DNA Complexes and Its Implication for Noninvasive Topical DNA Delivery to Skin

Arginine-rich cell penetrating peptides are powerful tools for in vitro as well as in vivo delivery of a wide plethora of biomolecules. However, presence of consecutive arginine residues leads to enhanced amenability for proteolytic degradation as well as steric hindrances for membrane interactions which compromise its bioavailability. In order to overcome these limitations we previously reported a safe and stable octaarginine based oligomer, i.e., (r-x-r)4-carbamate, where the backbone amide linkages were replaced by carbamate linkages and 6-aminohexanoic acid based spacer moieties were incorporated for better flexibility, hydrophobicity, optimal spacing of guanidinium groups, and protection against proteolytic cleavage; resulting in improved transfection efficiency over its amide counterpart. In the present work we have investigated the mechanism behind this enhanced transfection efficiency and, based on our observations, demonstrate how the synergistic effect of rationalized oligomer designing, complex characteristics, and cell type contributes to overall effective intracellular delivery. Our results indicate that the (r-x-r)4-carbamate-plasmid DNA complexes primarily utilize lipid raft dependent pathway of cellular entry more than other pathways, and this possibly facilitates their increased entry in the lipid raft rich milieu of skin cells. We also emphasize the utility of oligomer (r-x-r)4-carbamate as an efficient carrier for topical delivery of nucleic acids in skin tissue. This carrier can be utilized for safe, efficient, and noninvasive delivery of therapeutically relevant macromolecular hydrophilic cargo like nucleic acids to skin.

Redox-dependent Condensation of the Mycobacterial Nucleoid by WhiB4

Oxidative stress response in bacteria is mediated through coordination between the regulators of oxidant-remediation systems (e.g. OxyR, SoxR) and nucleoid condensation (e.g. Dps, Fis). However, these genetic factors are either absent or rendered non-functional in the human pathogen Mycobacterium tuberculosis (Mtb). Therefore, how Mtb organizes genome architecture and regulates gene expression to counterbalance oxidative imbalance is unknown. Here, we report that an intracellular redox-sensor, WhiB4, dynamically links genome condensation and oxidative stress response in Mtb. Disruption of WhiB4 affects the expression of genes involved in maintaining redox homeostasis, central metabolism, and respiration under oxidative stress. Notably, disulfide-linked oligomerization of WhiB4 in response to oxidative stress activates the protein’s ability to condense DNA. Further, overexpression of WhiB4 led to hypercondensation of nucleoids, redox imbalance and increased susceptibility to oxidative stress, whereas WhiB4 disruption reversed this effect. In accordance with the findings in vitro, ChIP-Seq data demonstrated non-specific binding of WhiB4 to GC-rich regions of the Mtb genome. Lastly, data indicate that WhiB4 deletion affected the expression of ~ 30% of genes preferentially bound by the protein, suggesting both direct and indirect effects on gene expression. We propose that WhiB4 structurally couples Mtb’s response to oxidative stress with genome organization and transcription.

157. Breaching the Barrier: Topical Delivery of Peptide-Based Nanocomplexes in Skin

Skin is a dynamic organ known for its protective functions. With a wide range of associated deblitating and untreatable conditions it has been explored for topical delivery of therapeutics for sustainable effects. In spite of the obvious advantages of the organ delivery of various hydrophilic molecules to skin has met with limited success. This could be attributed to the unique lipid composition and compact organization of stratum corneum which impedes the entry of such molecules in skin limiting their potential clinical translations. Recent approaches involve use of minimal invasive methods for macromolecules delivery to skin. However, issues with efficiency, toxicity, robustness and high costs limit their universal use. Thus one of the key challenges in skin biology is to develop noninvasive, non-toxic and efficient methods for delivery of biomolecules to and through the skin. We have developed a peptide-based delivery system for efficient nucleic acid delivery in skin upon topical application as nanocomplexes. Further we have tried to improvise the delivery efficiency using safe enhancers and modified the carrier to attain specific targeting in skin. The peptide is secondary amphipathic in nature that tends to acquire alpha-helical structure in hydrophobic environment and retains in skin till 24hrs as seen through Franz assay. We have also found that upon topical application of peptide either in bare form or as nanocomplex to skin cells or human foreskin tissue it exhibits efficient cellular entry, high transfection efficiency as well as skin penetration ability as assessed by fluorescence and luciferase based assays. Transfection efficiency observed was equivalent to that obtained with commercial agents. In-vivo studies using SKH-1 hairless mice model showed similar activity. To realize the clinical potential of the work we have shown delivery of therapeutically relevant nucleic acids in skin with effective and traceable amounts of therapeutic molecules. The cytotoxicity and dye penetration test analysis of bare peptide and nanocomplex revealed no deleterious effect on skin cells as well as tissue. Mechanistic insights revealed that the entry of the peptide in skin is mediated partially by fluidization of lipids in addition to transient disruption of junctional proteins as seen through FTIR and time lapse studies. To further enhance the transfection efficiency of these nanocomplexes in skin without compromising its integrity, pre-application of silicone oil worked as an effective strategy. We further modified the peptide with a keratinocyte specific ligand and observed high transfection efficiency in selective cellular population. Presently we are validating the same in skin tissue. Overall we describe development of a convenient, clinically effective and possibly patient compliant approach to facilitate delivery of macromolecular therapeutics in skin.

494. Glycosaminoglycans in Gene Delivery: An Effective Strategy for Enhancement of Transfection Efficiency of Amphipathic Peptides for Localized and Systemic Applications?

Glycosaminoglycans (GAGs) are negatively charged, linear, sulphated polysaccharides. Cell surface glycosaminoglycans have often been described as portals of cellular entry of cationic peptides and polymers used for gene delivery. On the other hand, exogenous glycosaminoglycans have generally been considered as impediments towards DNA delivery mediated through cationic vectors since they are likely to destabilize these electrostatically assembled complexes. However, recent reports have shown that exogenous glycosaminoglycans can also aid DNA delivery by reducing cytotoxicity and enhancing cellular uptake in some cationic polymers. Our laboratory has shown that addition of small amounts of exogenous GAGs to different cationic arginine-rich peptide-DNA nanocomplexes leads to an increase in their gene delivery efficiency. We observed formation of a ‘GAG coat’ on the nanocomplex surface which improved extracellular stability and subsequent cellular entry along with improved endosomal release and enhanced accumulation near the nucleus. However very little work has been done to understand the role of exogenous glycosaminoglycans on gene delivery by amphipathic peptides. We have developed a set of amphipathic peptides which exhibit high transfection efficiency in multiple cell lines. We explored the effect of addition of chondroitin sulphate to the peptide-DNA nanocomplexes. We observed an increase in nanocomplex size, decrease in surface charge and stability towards nuclease degradation at wt/wt ratios of 0.25 of chondroitin sulfate/peptide indicating its shielding effect. Enhanced transfection efficiency was also observed in multiple cell lines. Interestingly, maximum increase was observed in human keratinocyte cell line. Preliminary transfection results indicate that presence of CD44 could act as primary binding site for coated nanocomplexes leading to increased internalization. In addition, transfection in presence of inhibitors like Bafilomycin A1 indicates that the endosomal escape property of chondroitin sulphate might also play a part. This is being further verified through colocalization studies in presence of endosomal and lysosomal markers. Moreover, on topical application to human skin tissue, we observed increase in DNA delivery with the coated nanocomplexes. Since the skin tissue is also rich in glycosaminoglycans, we are exploring how the skin architecture might be helpful in aiding DNA delivery by GAG-coated nanocomplexes. Interestingly, while the transfection efficiency of the native nanocomplexes was compromised in presence of serum, addition of chondroitin sulfate was able to restore the transfection efficiency. Although the cellular uptake of the coated nanocomplexes was almost equal to that of the uncoated ones in presence of serum, the endosomal escape of the nanocomplexes was higher in coated nanocomplexes which could contribute towards the overall enhancement in transfection. All these results reveal the potential use of glycosaminoglycans to improve the transfection efficiency of amphipathic peptides for localized applications in skin as well as in vivo applications.

159. RXR-Carbamate – A Novel Molecular Transporter for Skin

Cell penetrating peptides are powerful tools for delivery of various therapeutics. One of the critical intrinsic features of these carriers is the presence of minimum 7-9 arginine residues which allows electrostatic interaction with cargos like nucleic acids as well as negatively charged cell surface molecules for facile entry and cargo delivery. However cellular entry might be compromised if the guanidium groups are consecutive and not spaced because of compromised cellular interaction. Another limitation is the amenability of these homoarginine systems to proteolytic cleavage which leads to their low bio-availability. We previously reported a safe and stable homoarginine peptide-based system (R-X-R)-carbamate where amide linkages were replaced by stable carbamate linkages and arginine residues were separated by aminohexanoic acid spacers. This alteration imparted better flexibility and hydrophobicity to these systems in addition to protection against proteolytic cleavage and minimal steric hindrances. These molecules entered cells efficiently and showed high transfection efficiencies in comparison to their amide counterparts. We have now investigated the mechanism behind enhanced transfection efficiency and based on our observations, demonstrate the applicability of this system as an efficient carrier for topical delivery of biomolecules in skin. We demonstrated through flow cytometry studies using chemical inhibitors of different known cellular uptake pathways that (R-X-R)-carbamate- DNA complexes not only show much higher uptake than amide counterpart but the uptake was drastically decreased in presence of inhibitor Methyl-β-Cyclodextrin -a known blocker of lipid raft mediated entry pathway. We have further validated the entry of these carriers using colocalization studies with pathway specific markers. Further, retention, stability and transfection efficiency studies involving depletion of cholesterol (which is a major component of lipid raft structures) using extraction method is being carried out. Comparative transfection efficiency in lipid raft enriched cancerous cell lines is being used for further validation. Lipid rafts act as major mediators of viral infections and are also involved in entry of nanocomplexes in skin. Therefore we explored if the (R-X-R)-carbamate-DNA complexes that are more biased to enter the cells through lipid raft dependant pathways also exhibit efficient entry into skin cells and tissue. The uptake of (R-X-R)-carbamate-DNA complexes and the transfection efficiency were studied in HaCaT cells and human skin tissue. High levels of transfection was achieved in human skin tissue after topical application of these complexes. Further studies on mechanism of interaction of these complexes with skin and the impact on skin integrity is currently ongoing. Ultimately this study will help us in obtaining a carrier system which is safe, more stable and efficient to deliver multiple cargo types in skin in a non-invasive manner for therapeutic applications. Moreover we intend to gain insights into the exact mechanism that it follows during its trajectory inside the skin cells and tissue.