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New Gene Therapy Reverses Atherosclerosis In Mice

Repair Biotechnologies, a company based in Syracuse, New York, has announced findings from early research suggesting that its technology can quickly stop the advancement of atherosclerosis. While these preclinical results are in mice, this approach has the potential for treating atherosclerosis in humans.

In March, the company announced that it had received positive feedback from the FDA regarding its pre-IND application. The company is now preparing for additional discussions as it moves forward with its plans for clinical trials.

A possible solution for atherosclerosis and familial hypercholesterolemia

Atherosclerosis occurs when plaque builds up in arteries, causing blockages that can result in heart attacks or strokes. It is the underlying cause of about 50% of all deaths in western society. Therefore, Repair Biotechnologies is using new technology to safely remove excess cholesterol in cells. This provides a different way to treat atherosclerosis, heart disease, and other conditions.

The company is developing lipid nanoparticles (LNPs) and messenger RNA (mRNA) to address various diseases. Its therapy focuses on reducing harmful cholesterol inside cells, not just in the blood. This can help prevent health issues caused by high cholesterol levels.

The company’s approach also shows promise in treating familial hypercholesterolemia, a genetic disorder in which the liver is less effective in removing excess LDL cholesterol. This results in elevated LDL levels in the bloodstream, posing health risks.

Preclinical results show promise

Researchers tested the LNP-mRNA treatment on mouse models of atherosclerosis and familial hypercholesterolemia for six weeks. Both test groups of mice showed a significant reduction in plaque formation. The company reported the following exciting results:

• Atherosclerotic mice saw a 19% reduction in plaque lipids and a 23% increase in plaque collagen. This is suggestive of artery plaque having stabilized.
• Familial hypercholesterolemia model mice saw a 17% reduction in plaque obstruction in the aortic root. Cardiovascular performance improved as well, with increased treadmill capacity.

We had the opportunity to talk to Reason, the CEO of Repair Biotechnologies, about these exciting results. He has long been an advocate for aging and rejuvenation research, which he has done for a long time on the blog Fight Aging.

Could you explain how your LNP and mRNA system works and how it degrades harmful cholesterol and reduces plaques?

Lipid nanoparticles (LNPs) are what they sound like, a tiny assembly of lipid molecules. Most LNPs used in therapy are in the 50 to 300 nanometer range. There are an infinite number of lipid combinations one could create. Only a few of these are useful, but those few are good at encapsulating materials such as small molecules, proteins, and nucleic acids and then introducing those materials into the cytosol of the cell.

The main thrust of LNP development in recent decades has been the production of safer LNPs, allowing higher and repeated dosing, and LNPs that have a greater ability to selectively reach specific tissues in the body.

If one is going to be introducing material into the cell cytoplasm, messenger RNA (mRNA) is a good candidate, as that is where mRNA needs to be in order for translation to proteins to take place in the ribosome. In the translation process, proteins are produced from the mRNA, and one mRNA molecule leads to the manufacture of many proteins before it is broken down.

Following COVID-19, the manufacture of mRNA has grown to become a sizable concern, and an entire industry is now tasked with figuring out how to make synthesized mRNA ever safer, more efficient, and non-immunogenic.

We presently use this system of LNP-delivered synthetic mRNA to generate our proprietary Cholesterol Degrading Platform (CDP) fusion protein inside cells in the liver. The liver is an easy target for all forms of drug that are injected intravenously, and many of the existing, better-known LNPs were developed specifically to target the liver. CDP consists of a number of proteins that are not normally expressed in conjunction, but when acting together, they target only excess free cholesterol molecules for degradation.

It is important to remember that cholesterol is usually modified in the body. It is either stashed in a cell membrane, esterified in droplets for storage inside cells, or attached to some form of lipoprotein particle for transport. Free cholesterol, the unmodified form, is toxic to cells. The presence of free cholesterol and consequent cell dysfunction and cell death contributes meaningfully to atherosclerosis, metabolic dysfunction-associated steatohepatitis (MASH), and a wide range of other conditions.

The options for free cholesterol clearance are limited by the points that (a) cholesterol is everywhere in cells and tissues and (b) our cells have no ability to break down excess cholesterol. There is no preexisting “break down excess cholesterol” process with a regulatory system that can be influenced by suitably designed small molecule drugs. The alternative possibility of small molecules that are designed to bind to and sequester cholesterol would kill cells by pulling the cholesterol from their membranes long before reaching a dose that is able to remove enough free cholesterol from inside cells to matter. The only path forward is to craft a sophisticated and selective assembly of protein machinery and deliver that assembly as a gene therapy, as we have done.

You had some really interesting results from the preclinical testing. Can you talk us through the findings and their implications?

To date, we have demonstrated rapid and profound reversal of disease in mouse models of (a) MASH, a progression of fatty liver towards liver failure that is characterized by fibrosis and loss of liver function, (b) atherosclerosis, the buildup of fatty plaques in blood vessel walls, leading to cardiovascular disease and stroke, and (c) homozygous familial hypercholesterolemia (HoFH), an inherited condition involving loss-of-function mutations in low-density lipoprotein receptors (LDLR) that causes high blood cholesterol and greatly accelerated atherosclerosis.

These three conditions are characterized by being largely irreversible under the present standards of care. While slowing the progression of disease is sometimes possible, few patients have been shown to achieve any meaningful reversal of established liver fibrosis or arterial atherosclerotic plaque, and the methods used to treat those patients are not consistently effective in other patients.

In each case, 6 to 8 weeks of once-weekly injections of CDP therapy produced sizable improvements in blood chemistry, including reductions in alanine aminotransferase (ALT), a measure of liver cell death and stress, and in histological assessments of disease. In MASH model mice, a 52% reduction in liver fibrosis was observed versus untreated controls.

In the ApoE-knockout mouse model of atherosclerosis, plaque lipids were reduced by 19% while plaque collagen increased by 23% versus controls, a dramatic stabilization of unstable plaques at risk of rupture. In the LDLR-knockout mouse model of HoFH, plaque cross-sectional area decreased by 17% and mouse treadmill performance improved by 60% versus controls, a considerable improvement in cardiovascular function.

To compare this with other present efforts, the drug, resmetirom (Madrigal Pharmaceuticals), recently approved by the FDA for the treatment of MASH, has no effect on fibrosis in mice over 8 weeks of treatment. In the MAESTRO human trial in patients with comparatively mild MASH, the treated groups saw only 25% reduction in fibrosis compared to 14% in the placebo group after 52 weeks of treatment.

In the case of atherosclerosis, large clinical trials have shown that long-term treatment with statins or other low-density lipoprotein (LDL)-lowering technologies such as PCSK9 inhibitors fails to produce a reduction in atherosclerotic plaque volume of more than a few percentage points. Our CDP therapy far outperforms these approaches to treatment.

Perhaps the most interesting outcome is that we have demonstrated that a localized excess of free cholesterol is indeed a major factor in many conditions, age-related and obesity-related. It had been theorized that this was the case for liver diseases such as MASH, but lacking a technology that selectively cleared only free cholesterol, this had to remain only a compelling theory. Armed with that selective clearance technology, our results have now convincingly demonstrated that free cholesterol is a major, important target for many conditions.

Let’s touch upon the scalability of this technology. A concern for many people interested in our field is access and affordability. Can you speak about if or how your technology is or is not scalable to address these concerns?

LNPs are very cheap to manufacture at scale. Still, LNP-mRNA is presently a comparatively expensive technology for development purposes because of the cost of producing synthetic mRNA. But synthetic mRNA has now had its fifteen minutes of fame in the context of COVID-19 vaccines, and there exist other therapies that use a great deal more mRNA per dose than a vaccine requires.

The incentive to reduce manufacturing costs is there, and work progresses on that front. If one looks at what has happened to the techniques and price of manufacture of adeno-associated virus (AAV) in recent decades, there have been improvements even in the absence of a mass-market AAV drug. Given a mRNA drug, such as ours, that can in principle be of use to most older people, the incentive to find ways to reduce manufacturing costs will be sizable.

Given the success of mRNA vaccines for COVID and how rapidly they were developed and distributed, what do you think our field can learn from that?

That regulatory caution depends on contexts other than utilitarian cost-benefit calculations. One can certainly look at the unmet need of atherosclerosis (larger) versus the unmet need of COVID-19 (smaller) and ask why regulators treat these two problems with the opposite degree of enthusiasm for the approval of therapies. But this misses the point about the way in which people think about the status quo versus new problems.

Your LNP/mRNA approach could have broader applications. I know some companies for example are exploring using mRNAs for senolytics and partial reprogramming. What other directions are you considering at Repair?

LNP-mRNA therapies are the most small-molecule-like of the gene therapies. They are delivered, have an effect for a few days, and then are no longer present in the patient. That makes it easy for conservative organizations, whether regulators or inventors, to fit them into their understanding of the world of medical development.

In terms of what comes next for Repair, there are so very many potential uses for CDP that it is hard to say which will rise to the top of the list after atherosclerosis and MASH. Recall that a number of common neurodegenerative conditions are characterized by dysregulated lipid metabolism and lipid droplets in misbehaving cells, for example. The use of CDP to treat any one of those, and the development of an appropriate LNP vector, could be a company-sized endeavor in and of itself.

What are the next steps for Repair Bio and moving towards clinical trials?

Raising and spending a great deal of funding! Once past our present point of conducting pre-IND meetings with the FDA, the heavily regulated path to a Biologic License Application (BLA) is an expensive proposition: good laboratory practice (GLP) studies in mice and non-human primates; setting up a good manufacturing practice (GMP) process for drug manufacture with a contract development and manufacturing organization (CDMO); manufacturing the GMP-grade drug for animal and human studies; organizing a clinical trial and engaging a specialist contract research organization (CRO) to run it; and all of the other necessary high-cost parts of the puzzle.

Switching tack for a moment: only a decade ago, the biggest problem for our field was funding for early-stage research, which seems to have improved in recent years. What would you say is now the greatest bottleneck in our field to getting rejuvenation biotechnologies to the masses?

I would say that there are two biggest problems. The first is that there remains too little funding for optimal progress, both for research and later development. Once a program makes the leap from academia to a biotech startup, these companies are largely finding seed-stage funding, at least when the overall financial markets are in decent shape.

Once a company is at the Series A or pre-IND stage, between proof of concept and first clinical trial, it becomes much harder to find funding. It is too far along for the early stage investors, and not far enough along for the institutional biotech investors with deep pockets.

The second biggest problem is there there is no infrastructure to bring generic drugs that we believe are likely useful (e.g. rapamycin or the senolytic dasatinib and quercetin combination) or non-drug procedures that we believe are likely useful (e.g. fecal microbiota transplantation or many forms of stem cell transplantation still only accessible via medical tourism) into widespread use.

There is no panoply of philanthropic organizations set up to run the necessary clinical trials to convince physicians that these interventions are in fact great. Those trials could run at a much lower cost than the sort of trial required by the FDA, lacking most of the expensive frills, but still aiming at responsible, robust creation of human data.

It has been nearly a decade since the first mouse data on dasatinib and quercetin was published, and five years since a human trial confirmed this treatment to reduce the burden of senescent cells in patients. Yet only a small number of other clinical trials are underway, and other than a few self-experimenters, and the patients of a few anti-aging physicians, older people are not using this general, off-label drug combination.

There is no energetic effort to assess efficacy in clinical trials for more than just a few of the large number of age-related conditions that clearance of senescent cells has been shown to improve in mice. It seems madness for this situation of little data, little usage, and few attempts to produce that data to continue. Yet here we are.

As a long-standing advocate for our field, how do you think things have changed in the last decade and are you more optimistic or pessimistic about the future?

Pessimistic in the short term (timescales of a decade), optimistic in the long term (timescales of two decades and longer). There is a great deal of promising work underway now in academia and industry, far more than was the case a decade past, and tackling many more of the different aspects of aging.

That said, it is taking far too long for existing low-cost, generic approaches to incremental, piecemeal rejuvenation, such as the aforementioned fecal microbiota transplantation and dasatinib and quercetin combination, to be validated in a way that grasps the attention of our society at large and thereby enables widespread use.

Anything else you would like to share with our readers?

That you can make a difference. Twenty years ago, a small number of people, a few hundred at most, started the avalanche that led to today’s longevity industry. Some were scientists, some advocates, some ordinary folk who made a small donation to help the non-profits and research programs that started the ball rolling. Find a cause: every incremental act to help will produce an ever greater payoff over time.