The advent of genome editing has transformed the therapeutic landscape for several debilitating diseases, and the clinical outlook for gene therapeutics has never been more promising. The therapeutic ...potential of nucleic acids has been limited by a reliance on engineered viral vectors for delivery. Chemically defined polymers can remediate technological, regulatory, and clinical challenges associated with viral modes of gene delivery. Because of their scalability, versatility, and exquisite tunability, polymers are ideal biomaterial platforms for delivering nucleic acid payloads efficiently while minimizing immune response and cellular toxicity. While polymeric gene delivery has progressed significantly in the past four decades, clinical translation of polymeric vehicles faces several formidable challenges. The aim of our Account is to illustrate diverse concepts in designing polymeric vectors towards meeting therapeutic goals of in vivo and ex vivo gene therapy. Here, we highlight several classes of polymers employed in gene delivery and summarize the recent work on understanding the contributions of chemical and architectural design parameters. We touch upon characterization methods used to visualize and understand events transpiring at the interfaces between polymer, nucleic acids, and the physiological environment. We conclude that interdisciplinary approaches and methodologies motivated by fundamental questions are key to designing high-performing polymeric vehicles for gene therapy.
Quinine's ability to bind DNA and potentially inhibit transcription and translation has been examined as a mode of action for its antimalarial activity. UV absorption and fluorescence-based studies ...have lacked the chemical specificity to develop an unambiguous molecular-level picture of the binding interaction. To address this, we use Raman spectroscopy and molecular dynamics (MD) to investigate quinine-DNA interactions. We demonstrate that quinine's strongest Raman band in the fingerprint region, which derives from a symmetric stretching mode of the quinoline ring, is highly sensitive to the local chemical environment and pH. The frequency shifts observed for this mode in solvents of varying polarity can be explained in terms of the Stark effect using a simple Onsager solvation model, indicating that the vibration reports on the local electrostatic environment. However, specific chemical interactions between the quinoline ring and its environment, such as hydrogen bonding and π-stacking, perturb the frequency of this mode in a more complicated but predictable manner. We use this vibration as a spectroscopic probe to investigate the binding interaction between quinine and DNA. We find that, when the quinoline ring is protonated, quinine weakly intercalates into DNA by forming π-stacking interactions with the base pairs. The Raman spectra indicate that quinine can intercalate into DNA with a ratio reaching up to roughly one molecule per 25 base pairs. Our results are confirmed by MD simulations, which also show that the quinoline ring adopts a t-shaped π-stacking geometry with the DNA base pairs, whereas the quinuclidine head group weakly interacts with the phosphate backbone in the minor groove. We expect that the spectral correlations determined here will enable future studies to probe quinine's antimalarial activities, such as disrupting hemozoin biocrystallization, which is hypothesized to be, among other things, one of its primary modes of action against Plasmodium parasites.
Polymeric vehicles that efficiently package and controllably release nucleic acids enable the development of safer and more efficacious strategies in genetic and polynucleotide therapies. Developing ...delivery platforms that endogenously monitor the molecular interactions, which facilitate binding and release of nucleic acids in cells, would aid in the rational design of more effective vectors for clinical applications. Here, we report the facile synthesis of a copolymer containing quinine and 2-hydroxyethyl acrylate that effectively compacts plasmid DNA (pDNA) through electrostatic binding and intercalation. This polymer system poly(quinine-co-HEA) packages pDNA and shows exceptional cellular internalization, transgene expression, and low cytotoxicity compared to commercial controls for several human cell lines, including HeLa, HEK 293T, K562, and keratinocytes (N/TERTs). Using quinine as an endogenous reporter for pDNA intercalation, Raman imaging revealed that proteins inside cells facilitate the unpackaging of polymer–DNA complexes (polyplexes) and the release of their cargo. Our work showcases the ability of this quinine copolymer reporter to not only facilitate effective gene delivery but also enable diagnostic monitoring of polymer–pDNA binding interactions on the molecular scale via Raman imaging. The use of Raman chemical imaging in the field of gene delivery yields unprecedented insight into the unpackaging behavior of polyplexes in cells and provides a methodology to assess and design more efficient delivery vehicles for gene-based therapies.
After decades of development, gene therapy has finally reached the forefront of medicine and has led to new cures for genetic disorders and the development of life-saving vaccines. The field has been ...buoyed by the development of more precise and user-friendly targeted nucleases, such as those used for clustered regularly interspersed palindromic repeats (CRISPR)-based editing. These useful gene-editing technologies, however, are still stymied by the challenge of delivering exogenous nucleic acids and proteins into the cells of interest. The emerging gene therapy industry is investing heavily in developing more efficient and safe non-viral vehicles as alternatives to costly and immunogenic viral vectors. Cationic polymers are promising non-viral vectors due to their manufacturing scalability, their chemical stability, and their synthetic tunability. Improvements in delivery efficiency are necessary, however, for widespread adoption of polymeric vehicles for gene therapy. One challenge in improving performance, however, is the difficulty and limited methodology for elucidating the intracellular mechanics of polymeric vehicles. In this thesis, I describe my research focused on the development of a novel quinine-containing polymer, called a Quinine Copolymer Reporter (QCR), that enhanced transient transfections of cultured cells with plasmids and improved gene editing of cultured cells through the simultaneous delivery of the CRISPR-associated protein Cas9 and DNA donor template. In addition, I describe collaborative research performed with colleagues in the research group of Prof. Renee Frontiera that characterized a band in quinine’s Raman spectrum that is diagnostic of its chemical environment. Using this chemical sensitivity in conjunction with Raman microscopic imaging, we help elucidated the intracellular unpackaging mechanisms of the QCR-nucleic acid complexes.
Quinine is a promising natural product building block for polymer-based nucleic acid delivery vehicles as its structure enables DNA binding through both intercalation and electrostatic interactions. ...However, studies exploring the potential of quinine-based polymers for nucleic acid delivery applications (transfection) are limited. In this work, we used a hydroquinine-functionalized monomer, HQ, with 2-hydroxyethyl acrylate to create a family of seven polymers (HQ-X, X = mole percentage of HQ), with mole percentages of HQ ranging from 12 to 100%. We developed a flow cytometer-based assay for studying the polymer–pDNA complexes (polyplex particles) directly and demonstrate that polymer composition and monomer structure influence polyplex characteristics such as the pDNA loading and the extent of adsorption of serum proteins on polyplex particles. Biological delivery experiments revealed that maximum transgene expression, outperforming commercial controls, was achieved with HQ-25 and HQ-35 as these two variants sustained gene expression over 96 h. HQ-44, HQ-60, and HQ-100 were not successful in inducing transgene expression, despite being able to deliver pDNA into the cells, highlighting that the release of pDNA is likely the bottleneck in transfection for polymers with higher HQ content. Using confocal imaging, we quantified the extent of colocalization between pDNA and lysosomes, proving the remarkable endosomal escape capabilities of the HQ-X polymers. Overall, this study demonstrates the advantages of HQ-X polymers as well as provides guiding principles for improving the monomer structure and polymer composition, supporting the development of the next generation of polymer-based nucleic acid delivery vehicles harnessing the power of natural products.
Occupational therapy is underpinned by the premise that engagement in occupation is fundamental to health and well-being. Through occupations, people are able to orchestrate their lives in ways that ...enable them not only to survive, but also to experience human flourishing. Through occupation, people can develop and maintain their families, neighbourhoods and communities as sources of belonging, opportunities and common action. Occupation, therefore, is not only important to each individual, but also, through collective occupation, people develop the kind of lives that they live together. Occupation is an essential factor in life quality, the experience of being human and the social transformation of individuals and of the societies of which people are a part. This article describes the formation of an International Think Tank for Occupation based social transformation. It begins with a brief overview of the conditions and context that underpinned the development of the group, and the milestones achieved to date in the establishment of a global network.
DNA-based immunization strategies designed to elicit cellular antitumor immunity offer an attractive alternative to protein- or peptide-based approaches. In the present study we have evaluated the ...feasibility of DNA vaccination for the induction of CTL reactivity to five different melanoma Ags in vitro. Cultured, monocyte-derived dendritic cells (DC) were transiently transfected with plasmid DNA encoding human MART-1/Melan-A, pMel-17/gp100, tyrosinase, MAGE-1, or MAGE-3 by particle bombardment and used to stimulate autologous PBMC responder T cells. CTL reactivity to these previously identified melanoma Ags was reproducibly generated after two or three stimulations with genetically modified DC. Co-ordinate transfection of two melanoma Ag cDNAs into DC promoted CTL responders capable of recognizing epitopes from both gene products. Coinsertion of genes encoding the Th1-biasing cytokines IL-12 or IFN-alpha consistently enhanced the magnitude of the resulting Ag-specific CTL reactivity. Importantly, DC transfected with a single melanoma Ag cDNA were capable of stimulating Ag-specific CTL reactivity restricted by multiple host MHC alleles, some of which had not been previously identified. These results support the inherent strengths of gene-based vaccine approaches that do not require prior knowledge of responder MHC haplotypes or of relevant MHC-restricted peptide epitopes. Given previous observations of in situ tumor HLA allele-loss variants, DC gene vaccine strategies may elicit a greater diversity of host therapeutic immunity, thereby enhancing the clinical utility and success of such approaches.
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