Periodic Reporting for period 1 - GETPolPhys (Gene Electro-Transfer Through the Lens of Polymer Physics)
Reporting period: 2021-06-01 to 2023-05-31
Genes, or snippets of DNA, are instructions that are read by cells in our body to produce proteins that carry out a plethora of life's essential functions. Gene therapy relies on modifying defective genes or introducing new genes, thereby restoring functionality or introducing new functionality. Gene therapy offers a therapeutic potential against a wide variety of ailments ranging from genetic diseases to cancer and infectious diseases. One of the major hurdles of gene therapy is gene delivery; DNA molecules carrying the gene cannot cross the formidable barrier of the cell membrane and enter cells to exert its therapeutic potential. Methods of introducing genes to cells, that are both efficient and safe, are needed. Electroporation is one such method that relies on applying an electric field to transiently permeabilize the cell membrane and introduce foreign molecules, including genes, to cells. Although safe, electroporation is not efficient at introducing genes to cells in our body. This action - GETPolPhys or Gene Electro-Transfer through the lens of polymer physics - was aimed at developing a fundamental understanding of gene delivery using electroporation. Using the fundamental understanding, major barriers to electroporation mediated gene delivery (or gene electro-transfer) were identified and overcome.
Gene therapies have so far relied on viruses to deliver the genes to cells. A major problem of virus based gene therapy is safety; administering viruses leads to severe toxicity [1]. Another major problem is the cost; a virus based gene therapy for treatment of Spinal Muscular Atrophy costs around 2 millions euros [2]. The exorbitant costs of such therapies prevents its widespread adoption, especially amongst the low and middle income countries. Delivering chemotherapeutics using electroporation has proven to be safe, effective and easy to use [3]. Moreover, production of DNA molecules used in gene-electrotransfer has only 1/3rd the cost of virus production [4]. However, gene-electrotransfer is limited by its efficiency [5]. Improving the efficiency of gene-electrotransfer by fundamentally understanding the barriers will unleash the full potential of gene therapy in the clinics, allow its wide-spread adoption owing to its low cost of administration and offer a safer route to gene therapy.
The overall objective of this action was to obtain a fundamental understanding of how DNA molecules overcome the barriers of (a) the extracellular space and (b) the cell membrane during gene electro-transfer using principles of polymer physics and statistical mechanics. The use of these principles are indispensable to understanding the mechanisms of gene-electrotransfer, and an understanding based on these principles will lead to enhancing the efficiency of gene electro-transfer from first principles as opposed to the current time and resource intensive approach of trial-and-error. A parallel goal of the action was to also develop the skills of the fellow in the field of pre-clinical research and clinical aspects of gene therapies and advance his career in translation of therapies towards clinics.
[1] Wilson, J. M., & Flotte, T. R., Genetic Engineering & Biotechnology News 2020, 40(8), 14-16. [2] Nuijten, M., Journal of Market Access & Health Policy 2022, 10(1), 2022353. [3] Cemazar, M., & Sersa, G., Bioelectricity 2019, 1(4), 204-213. [4] Ran, T., Eichmüller et al., International journal of cancer 2020, 147(12), 3438-3445. [5] Sachdev, S. et al., Bioelectrochemistry 2022, 144, 107994.
Gene therapies have so far relied on viruses to deliver the genes to cells. A major problem of virus based gene therapy is safety; administering viruses leads to severe toxicity [1]. Another major problem is the cost; a virus based gene therapy for treatment of Spinal Muscular Atrophy costs around 2 millions euros [2]. The exorbitant costs of such therapies prevents its widespread adoption, especially amongst the low and middle income countries. Delivering chemotherapeutics using electroporation has proven to be safe, effective and easy to use [3]. Moreover, production of DNA molecules used in gene-electrotransfer has only 1/3rd the cost of virus production [4]. However, gene-electrotransfer is limited by its efficiency [5]. Improving the efficiency of gene-electrotransfer by fundamentally understanding the barriers will unleash the full potential of gene therapy in the clinics, allow its wide-spread adoption owing to its low cost of administration and offer a safer route to gene therapy.
The overall objective of this action was to obtain a fundamental understanding of how DNA molecules overcome the barriers of (a) the extracellular space and (b) the cell membrane during gene electro-transfer using principles of polymer physics and statistical mechanics. The use of these principles are indispensable to understanding the mechanisms of gene-electrotransfer, and an understanding based on these principles will lead to enhancing the efficiency of gene electro-transfer from first principles as opposed to the current time and resource intensive approach of trial-and-error. A parallel goal of the action was to also develop the skills of the fellow in the field of pre-clinical research and clinical aspects of gene therapies and advance his career in translation of therapies towards clinics.
[1] Wilson, J. M., & Flotte, T. R., Genetic Engineering & Biotechnology News 2020, 40(8), 14-16. [2] Nuijten, M., Journal of Market Access & Health Policy 2022, 10(1), 2022353. [3] Cemazar, M., & Sersa, G., Bioelectricity 2019, 1(4), 204-213. [4] Ran, T., Eichmüller et al., International journal of cancer 2020, 147(12), 3438-3445. [5] Sachdev, S. et al., Bioelectrochemistry 2022, 144, 107994.
The work in this action was conducted through five work packages (WPs). WP1-3 comprised of work conducted towards the research objectives, which were to address and overcome the barriers to gene electro-transfer. At the beginning of the action, a review article was published that highlighted and detailed the barriers to gene electro-transfer. The review article is currently being used by academic researchers and industry professionals who want to understand and advance the field of gene electro-transfer. Further, the fellow also gave an invited plenary talk at a conference, where gene electro-transfer was introduced to engineers outside the field with the aim of spreading awareness regarding the inter-disciplinary aspects of this action, and of biotechnology research in general. In WP1, the work done by the fellow resulted in an oral presentation at the World Congress on Electroporation. The conference was attended by more than 400 people from all around the world. In WP2, the work conducted in WP1 was extended through the secondment, and the fellow is currently working on a manuscript combining the works done in WP1 and WP2. WP3 comprised of work that yielded an additional conference presentation and 2 journal publications. The fellow also gave an invited faculty lecture related to works conducted in WP1-3 at an international workshop on Electroporation Based Technologies and Treatments (EBTT) organised by the host institution.
Throughout the action, the fellow indulged in training activities through WP4 that helped him develop from his chemical engineering background to pre-clinical research and clinical aspects of therapies. This also included work conducted at the Institute of Oncology Ljubljana, Slovenia (secondment) and completing an online course on cell therapies. WP5 comprised of management of the project and the dissemination of results obtained in the project.
Work conducted in this action is reported in: (1) a review article that highlights and details the barriers to gene electro-transfer, including a current understanding of how DNA molecules overcome these barriers; (2) forthcoming journal paper on how DNA migrates through extracellular barrier; (3) journal papers related to overcoming the cell membrane barrier.
Throughout the action, the fellow indulged in training activities through WP4 that helped him develop from his chemical engineering background to pre-clinical research and clinical aspects of therapies. This also included work conducted at the Institute of Oncology Ljubljana, Slovenia (secondment) and completing an online course on cell therapies. WP5 comprised of management of the project and the dissemination of results obtained in the project.
Work conducted in this action is reported in: (1) a review article that highlights and details the barriers to gene electro-transfer, including a current understanding of how DNA molecules overcome these barriers; (2) forthcoming journal paper on how DNA migrates through extracellular barrier; (3) journal papers related to overcoming the cell membrane barrier.
The action has pushed the boundaries of knowledge on gene-electrotransfer in many ways. The review article published during the action has provided a fresh perspective on the barriers that limit the efficiency of gene-electrotransfer. The perspectives have been obtained using principles of polymer physics and statistical mechanics, not explored by the community so far, and are providing a basis to fundamentally understand gene-electrotransfer. The review has practical implications as well, as it is being used by industry professionals to venture into the field of gene electro-transfer.
The results of the action have also showed how DNA distributes in the tissue after injection, and what processes dominate the distribution of DNA in the tissue. Further, a method to control this distribution has also been demonstrated, which is an improvement over the current method of injecting DNA to tissues that leads to an uncontrolled distribution.
Further, gene electro-transfer has been achieved using electric field pulses of nano-second duration and an enhanced understanding of the forces that lead to DNA molecules coming in contact with the cell membrane has been developed. In addition, nano-channel gene electro-transfer has also been achieved using commercially available trans-well inserts that offer a cheaper alternative to expensive and difficult to manufacture devices used currently.
Impacts anticipated from the results of the action are enhanced understanding of the gene electro-transfer process which will yield efficient protocols in the clinics for patients and wider adoption of gene electro-transfer technique in research laboratories.
The results of the action have also showed how DNA distributes in the tissue after injection, and what processes dominate the distribution of DNA in the tissue. Further, a method to control this distribution has also been demonstrated, which is an improvement over the current method of injecting DNA to tissues that leads to an uncontrolled distribution.
Further, gene electro-transfer has been achieved using electric field pulses of nano-second duration and an enhanced understanding of the forces that lead to DNA molecules coming in contact with the cell membrane has been developed. In addition, nano-channel gene electro-transfer has also been achieved using commercially available trans-well inserts that offer a cheaper alternative to expensive and difficult to manufacture devices used currently.
Impacts anticipated from the results of the action are enhanced understanding of the gene electro-transfer process which will yield efficient protocols in the clinics for patients and wider adoption of gene electro-transfer technique in research laboratories.