CURIOUS FEBURARY 2021

Gene therapy strategy found effective in mouse model of hereditary

disease TSC
Patients with tuberous sclerosis complex, a genetic disorder characterized by the growth of noncancerous tumors in
multiple organs of the body, have limited treatment options. A team led by investigators at Massachusetts General
Hospital (MGH) has now shown that gene therapy can effectively treat mice that express one of the mutated genes
that cause the disease. The research is published in Science Advances.

The gene, called TSC2, codes for tuberin, a protein that acts to inhibit cell growth and proliferation. When mutations
occur in TSC2, resulting in a lack of tuberin in cells, the cells enlarge and multiply, leading to the formation of tumors.

To restore the function of TSC2 and tuberin in a mouse model of tuberous sclerosis complex, researchers developed
a form of gene therapy using an adeno-associated virus vector carrying the DNA that codes for a condensed form of
tuberin (which fits within the vector's carrying capacity) and functions like the normal full-length tuberin protein.

Mice with tuberous sclerosis complex had a shortened life span of about 58 days on average, and they showed signs
of brain abnormalities consistent with those that are often seen in patients with the disease. When the mice were
injected intravenously with the gene therapy treatment, however, their average survival was extended to 462 days,
and their brains showed reduced signs of damage.

“Current treatments for tuberous sclerosis complex include surgery and/or lifelong treatment with drugs that cause
immune suppression and potentially compromise early brain development. Therefore, there is a clear need to
identify other therapeutic approaches for this disease," says co-lead author Shilpa Prabhakar, an investigator in the
MGH departments of Neurology and Radiology. "Adeno-associated virus vectors have been used widely in clinical
trials for many hereditary diseases with little to no toxicity, long-term action in nondividing cells, and improvement
in symptoms," adds Prabhakar. She notes that benefits can be seen after a single injection, and some forms of the
viral vector can efficiently enter the brain and peripheral organs after intravenous injection.

The U.S. Food and Drug Administration has approved a limited number of gene therapy products for use in humans,
and the results from this study suggest that clinical trials are warranted to test the strategy's potential in patients with
tuberous sclerosis complex.

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 CURIOUS FEBURARY 2021

New way to deliver DNA-based therapies for diseases

Polymers could be used to create less expensive gene therapies or vaccines for diseases
University of Minnesota Twin Cities researchers in the Department of Chemistry have created a new polymer to
deliver DNA and RNA-based therapies for diseases. For the first time in the industry, the researchers were able to see
exactly how polymers interact with human cells when delivering medicines into the body. This discovery opens the
door for more widespread use of polymers in applications like gene therapy and vaccine development.

The research is published in the Proceedings of the National Academy of Sciences (PNAS).
Gene therapy involves altering the genes inside the body's cells to treat or cure diseases. It requires a carrier that
"packages" the DNA to deliver it into the cell -- oftentimes, a virus is used as a carrier. Packaging of nucleic acids is
also used in vaccines, such as the recently developed messenger RNA (mRNA) COVID-19 vaccine, which is enclosed
in a lipid.

The research team is led by chemistry professor Theresa Reineke and associate professor Renee Frontiera. Reineke’s
lab synthesizes polymers, which are long-chain molecules that make up plastics, to use for packaging the nucleic acids
instead.

"It's kind of like ordering something from Amazon, and it's shipped in a box," Reineke explained. "Things get broken
if they're not delivered in a package. That's basically what we're doing here but on a nano-level. We're taking these
really sensitive RNA and DNA cargo that are susceptible to enzymatic degradation, that won't get to their target unless
you have something to protect them."

The researchers designed the copolymer using quinine, a naturally occurring substance used in tonic water, and 2
hydroxyethyl acrylate (HEA), which makes the material soluble and is used in a variety of personal care and medical
materials. Because quinine is fluorescent, the research team was able to track the DNA package throughout the body
and into the cells using Raman spectroscopy, a chemical imaging technique.

"We've discovered a new packaging tool with this natural product that's important for all of these high-flying,
important fields like gene therapy and vaccines," said Reineke, who is also a Distinguished McKnight University
Professor. "And, it works in a variety of cell-types. On top of that, it's got all of these cool features -- it's fluorescent,
we can track it, it's Raman active, and that allowed us to understand a lot of fundamentals about these packaging
systems that were impossible to probe before we incorporated this natural product."

Polymer-based drug delivery is significantly cheaper than using viruses, especially for gene therapy, which can cost up
to $2 million for a single injection. However, the main barrier preventing widespread polymer use was that scientists
didn't know a lot about how the polymer package actually interacts with cells in the body.

This research helps clear up that uncertainty. Frontiera's lab specializes in chemical imaging. Using Raman
spectroscopy, they discovered that a cell's own proteins play a key role in unpacking the nucleic acid cargo once the
polymer carrier enters the cell.

"It's very satisfying to know how this is actually happening, what the process of delivery is, and to actually see that in
real-time," Frontiera said.

“A key point is that these polymers also work very well. For all the beneficial attributes,
they're also incredibly effective at getting the payload into cells, and we were able to tell why, which doesn't always
happen in this field."

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