electroporation

How Gene Therapy Works
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How Gene Therapy Works

We’re living in the incredible future, but gene therapy (which holds the potential to cure otherwise deadly diseases at their source) is still a highly experimental field. Tune in to learn how the classic techniques of gene alteration work — and don’t work.

We break down the principles behind cell microinjection (what it sounds like: really tiny needles), electroporation (using the electrical properties of cell membranes to guide new genetic material into place), and sonoporation (using ultrasound to both harness cell membranes’ electrical properties and simultaneously deliver new genetic material).

[Image courtesy the DOE Joint Genome Institute.]

And hey, this episode made Stitcher’s Brain-Food Fix list the week it was published. Thanks, Stitcher!

Polymer patches could replace needles and enable more effective DNA vaccines

A dissolvable polymer film that allows the vaccination needle to be replaced with a patch is being developed by an MIT team. This development will not only make vaccinations less harrowing, but also allow for developing and delivering vaccines for diseases too dangerous for conventional techniques.

Vaccines work by causing the body’s immune system to kick into action before a real threat of infection exists. When the body encounters an infection, such as a virus, it generates antibodies that are specific to that infection. If a person encounters the same infection again, the body tags the incoming microbes with the antibodies. A vaccine is a sort of artificial disease that tells the body what the genuine virus is like without, ideally, the peril of actual infection. When a vaccine is injected, the body creates antibodies that are specific to the virus, so if the person encounters it, the immune system is primed and ready. However, this approach can be too risky with certain viruses, including HIV.

In a paper appearing in an online issue of Nature Materials, MIT researchers describe a new type of vaccine-delivery film that holds promise for improving the effectiveness of DNA vaccines. If such vaccines could be successfully delivered to humans, they could overcome not only the safety risks of using viruses to vaccinate against diseases such as HIV, but they would also be more stable, making it possible to ship and store them at room temperature. This type of vaccine delivery would also eliminate the need to inject vaccines by syringe, says Darrell Irvine, an MIT professor of biological engineering and materials science and engineering. “You just apply the patch for a few minutes, take it off and it leaves behind these thin polymer films embedded in the skin,” he says. Irvine and Paula Hammond, the David H. Koch Professor in Engineering, are the senior authors of the Nature Materials paper. Both are members of MIT’s David H. Koch Institute for Integrative Cancer Research. The lead author of the paper is Peter DeMuth, a graduate student in biological engineering.

Scientists have had some recent success delivering DNA vaccines to human patients using a technique called electroporation. This method requires first injecting the DNA under the skin, then using electrodes to create an electric field that opens small pores in the membranes of cells in the skin, allowing DNA to get inside. However, the process can be painful and give varying results, Irvine says. “It’s showing some promise but it’s certainly not ideal and it’s not something you could imagine in a global prophylactic vaccine setting, especially in resource-poor countries,” he says. Irvine and Hammond took a different approach to delivering DNA to the skin, creating a patch made of many layers of polymers embedded with the DNA vaccine. These polymer films are implanted under the skin using microneedles that penetrate about half a millimeter into the skin — deep enough to deliver the DNA to immune cells in the epidermis, but not deep enough to cause pain in the nerve endings of the dermis. Once under the skin, the films degrade as they come in contact with water, releasing the vaccine over days or weeks. As the film breaks apart, the DNA strands become tangled up with pieces of the polymer, which protect the DNA and help it get inside cells. The researchers can control how much DNA gets delivered by tuning the number of polymer layers. They can also control the rate of delivery by altering how hydrophobic (water-fearing) the film is. DNA injected on its own is usually broken down very quickly, before the immune system can generate a memory response. When the DNA is released over time, the immune system has more time to interact with it, boosting the vaccine’s effectiveness. The polymer film also includes an adjuvant — a molecule that helps to boost the immune response. In this case, the adjuvant consists of strands of RNA that resemble viral RNA, which provokes inflammation and recruits immune cells to the area. The ability to provoke inflammation is one of the key advantages of the new delivery system. Other benefits include targeting the wealth of immune cells in the skin, the use of a biodegradable delivery material, and the possibility of pain-free vaccine delivery.

In tests with mice, the researchers found that the immune response induced by the DNA-delivering film was as good as or better than that achieved with electroporation. To test whether the vaccine might provoke a response in primates, the researchers applied a polymer film carrying DNA that codes for proteins from the simian form of HIV to macaque skin samples cultured in the lab. In skin treated with the film, DNA was easily detectable, while DNA injected alone was quickly broken down. The researchers now plan to perform further tests in non-human primates before undertaking possible tests in humans. If successful, the vaccine-delivering patch could potentially be used to deliver vaccines for many different diseases, because the DNA sequence can be easily swapped out depending on the disease being targeted. 

In vivo rapid gene delivery into postmitotic neocortical neurons using iontoporation.

PubMed: In vivo rapid gene delivery into postmitotic neocortical neurons using iontoporation.

Nat Protoc. 2015 Jan;10(1):25-32 

Authors: De la Rossa A, Jabaudon D

Abstract
This protocol describes a method for directing the expression of genes of interest into postmitotic neocortical neurons in vivo. Microinjection of a DNA plasmid-amphiphilic molecule mix into the neocortex followed by delivery of an ad hoc electric pulse protocol during the first few days of life in mice allows rapid, focal and efficient expression of genes in postmitotic neurons. Compared with other gene delivery techniques such as in utero electroporation and viral infection, this method allows rapid (12 h), focal (50-200 μm), mosaic-like (50 to several hundred neurons) targeting of postmitotic neurons within existing circuits. This ‘iontoporation’ protocol, which can be completed within ∼20 min per mouse, allows straightforward assessment of genetic constructs in postmitotic cortical neurons and subsequent genetic, histological and physiological investigations of gene function.

PMID: 25474030 [PubMed - as supplied by publisher] http://dlvr.it/7kfsdV