Virus World

Recombinant adeno-associated viral vectors (rAAVs) are among the most commonly used vehicles for in vivo based gene therapies. However, it is hard to predict which AAV capsid will provide the most robust expression in human subjects due to the observed discordance in vector-mediated transduction between species. In our study, we use a primate specific capsid, AAV-LK03, to demonstrate that the limitation of this capsid towards transduction of mouse cells is unrelated to cell entry and nuclear transport but rather due to depleted histone H3 chemical modifications related to active transcription, namely H3K4me3 and H3K27ac, on the vector DNA itself. A single-amino acid insertion into the AAV-LK03 capsid enables efficient transduction and the accumulation of active-related epigenetic marks on the vector chromatin in mouse without compromising transduction efficiency in human cells. Our study suggests that the capsid protein itself is involved in driving the epigenetic status of the vector genome, most likely during the process of uncoating. Programming viral chromatin states by capsid design may enable facile DNA transduction between vector and host species and ultimately lead to rational selection of AAV capsids for use in humans. rAAV vectors vary in their effectiveness between species, making it difficult to predict clinical outcomes. Here authors show that AAV capsid proteins influence the vector epigenomic state in cells, and that a single amino acid change in the vector can alter the vector epigenome and hence transgene expression levels between species.

Published in  Nat. Communications (April 17, 2023):

https://doi.org/10.1038/s41467-023-38106-3 

As people age, they tend to develop diseases such as heart failure, kidney failure, diabetes, and obesity, and the presence of any one disease increases the risk of developing others. Traditional drug treatments, however, each target one condition. That means patients often have to take multiple medications, increasing both the risk of negative side effects and the likelihood of forgetting one. New research from the Wyss Institute for Biologically Inspired Engineering at Harvard University and Harvard Medical School (HMS) suggests that it may be possible someday to tend to multiple ailments with one treatment.

In the Wyss study, a single administration of an adeno-associated virus (AAV)-based gene therapy, which delivered combinations of three longevity-associated genes to mice, dramatically improved or completely reversed multiple age-related diseases, suggesting that a systems-level approach to treating such diseases could improve overall health and lifespan. The research is reported in PNAS. “The results we saw were stunning and suggest that holistically addressing aging via gene therapy could be more effective than the piecemeal approach that currently exists,” said first author Noah Davidsohn, a former research scientist at the Wyss Institute and HMS who is now chief technology officer of Rejuvenate Bio. “Everyone wants to stay as healthy as possible for as long as possible, and this study is a first step toward reducing the suffering caused by debilitating diseases.”

The study was conducted in the lab of Wyss core faculty member George Church as part of Davidsohn’s postdoctoral research into the genetics of aging. Davidsohn, Church, and their co-authors homed in on three genes that had been shown to confer increased health and lifespan benefits in mice that were genetically engineered to overexpress them: FGF21, sTGFβR2, and αKlotho. They hypothesized that providing extra copies of those genes to nonengineered mice via gene therapy would similarly combat age-related diseases and bring health benefits. The team created separate gene therapy constructs for each gene using the AAV8 serotype as a delivery vehicle, and injected them into mouse models of obesity, Type 2 diabetes, heart failure, and renal failure both individually and in combination with the other genes to see whether there was a positive synergistic effect. 

FGF21 caused complete reversal of weight gain and Type 2 diabetes in obese, diabetic mice following a single gene therapy administration, and its combination with sTGFβR2 reduced kidney atrophy by 75 percent in mice with renal fibrosis. Heart function in mice with heart failure improved by 58 percent when they were given sTGFβR2 alone or in combination with either of the other two genes, showing that a combined therapeutic treatment of FGF21 and sTGFβR2 could successfully treat all four age-related conditions, therefore improving health and survival. Administering all three genes together resulted in slightly worse outcomes, likely from an adverse interaction between FGF21 and αKlotho, which remains to be studied. Importantly, the injected genes remained separate from the animals’ native genomes, did not modify their DNA, and could not be passed to future generations or between living animals. 

“Achieving these results in nontransgenic mice is a major step toward being able to develop this treatment into a therapy, and co-administering multiple disease-addressing genes could help alleviate the immune issues that could arise from the alternative of delivering multiple, separate gene therapies for each disease,” said Church, who is also a professor of genetics at HMS and professor of health sciences and technology at Harvard and MIT. “This research marks a milestone in being able to effectively treat the many diseases associated with aging, and perhaps could lead to a means of addressing aging itself.”

Published in P.N.A.S. (November 4, 2019):

https://doi.org/10.1073/pnas.1910073116

In experiments with rats, pigs and monkeys, Johns Hopkins Medicine researchers have developed a way to deliver sight-saving gene therapy to the retina. If proved safe and effective in humans, the technique could provide a new, more permanent therapeutic option for patients with common diseases such as wet age-related macular degeneration (AMD), and it could potentially replace defective genes in patients with inherited retinal disease.

The new approach, described in the Aug. 13 issue of The Journal of Clinical Investigation, uses a small needle to inject harmless, genetically engineered viruses into the space between the white of the eye and the eye's vascular layer, called the suprachoroidal space. From there, the virus can spread throughout the eye to deliver therapeutic genes to cells in the retina. The gene therapy approach currently used to treat Leber congenital amaurosis, an inherited eye disorder, involves a surgical procedure to inject the gene-carrying virus under the retina. This procedure carries a high risk of patients developing cataracts, and a low but significant risk of retinal detachment and other vision-threatening complications.

Though only tested in animals up to this point, the new suprachoroidal injection technique is less invasive because it does not involve detaching the retina, and theoretically, it could be done on an outpatient basis, marking a major step toward making permanent vision-saving gene therapies safer and more accessible. A subretinal injection gene therapy approach is also being tested in clinical trials for age-related macular degeneration, which is among the leading causes of irreversible and disabling vision loss in people over age 50, according to the National Eye Institute. An estimated 10 million Americans have age-related macular degeneration. In the disease's more common "wet" form, abnormal blood vessels grow under the retina and leak vision-blocking fluids into the eye. The growth and leakage of the abnormal blood vessels is caused by excess production of a cell signal called vascular endothelial growth factor (VEGF).

Currently, eye specialists can stave off vision loss by injecting a protein into the eye that blocks VEGF, but these treatments have a limited life span, so patients must return to the clinic every four to six weeks for more injections to maintain their vision. Missed appointments can allow the abnormal blood vessels to grow, causing further vision loss. "We find that repeated treatments, although effective, can be hard for patients to keep up with, and over time, they lose vision," says Campochiaro. However, a gene therapy could turn each cell in the retina into a little pharmaceutical factory that constantly produces anti-VEGF proteins, thereby continuously maintaining vision without repeated injections.....

Study published on August 13 in the Journal of Clinical Investigation:

https://doi.org/10.1172/JCI129085

Infectious disease researchers have used a gene editing approach to remove latent herpes simplex virus 1, or HSV-1, also known as oral herpes. In animal models, the findings show at least a 90 percent decrease in the latent virus, enough researchers expect that it will keep the infection from coming back. The study, published August 18 in Nature Communications, used two sets of genetic scissors to damage the virus's DNA, fine-tuned the delivery vehicle to the infected cells, and targeted the nerve pathways that connect the neck with the face and reach the tissue where the virus lies dormant in individuals with the infection. "This is the first time that scientists have been able to go in and actually eliminate most of the herpes in a body," said senior author Dr. Keith Jerome, professor in the Vaccine and Infectious Disease Division at Fred Hutch. "We are targeting the root cause of the infection: the infected cells where the virus lies dormant and are the seeds that give rise to repeat infections." Most research on herpes has focused on suppressing the recurrence of painful symptoms, and Jerome said that his team is taking a completely different approach by focusing on how to cure the disease. "The big jump here is from doing this in test tubes to doing this in an animal," said Jerome, who also leads the Virology Division at UW Medicine. "I hope this study changes the dialog around herpes research and opens up the idea that we can start thinking about cure, rather than just control of the virus."  Two-thirds of the world population under the age of 50 have HSV-1, according to the World Health Organization. The infection primarily causes cold sores and is lifelong.

In the study, the researchers used two types of genetic scissors to cut the DNA of the herpes virus. They found that when using just one pair of the scissors the virus DNA can be repaired in the infected cell. But by combining two scissors -- two sets of gene-cutting proteins called meganucleases that zero in on and cut a segment of herpes DNA -- the virus fell apart. "We use a dual meganuclease that targets two sites on the virus DNA," said first author Martine Aubert, a senior staff scientist at Fred Hutch. "When there are two cuts, the cells seem to say that the virus DNA is too damaged to be repaired and other molecular players come in to remove it from the cell body." The dual genetic scissors are introduced into the target cells by delivering the gene coding for the gene-cutting proteins with a vector, which is a harmless deactivated virus that can slip into infected cells. The researchers injected the delivery vector into a mouse model of HSV-1 infection, and it finds its way to the target cells after entering the nerve pathways.  The researchers found a 92% reduction in the virus DNA present in the superior cervical ganglia, the nerve tissue where the virus lies dormant. The reductions remained for at least a month after the treatment and is enough the researchers say to keep the virus from reactivating.

The team did other comparisons to fine-tune the gene editing approach:

- Gene cuts with meganucleases were more efficient that with CRISPR/Cas9.

- Refining the vector delivery mechanism, they found the adeno-associated virus (AAV) vector that was the most efficient at getting the gene edits to cells infected with the virus.

The researchers are pursuing a similar strategy for herpes simplex 2, which causes genital herpes. They expect it to take at least 3 years to move toward clinical trials. "This is a curative approach for both oral and genital HSV infection," Aubert said. "I see it going into clinical trials in the near future."

Original study published in NAt. Medicine (August 18, 2020):

https://doi.org/10.1038/s41467-020-17936-5

Teams of researchers from Charité – Universitätsmedizin Berlin and the Medical University of Innsbruck have developed a new therapeutic concept for the treatment of temporal lobe epilepsy. It represents a gene therapy capable of suppressing seizures at their site of origin on demand. Having been shown to be effective in an animal model, the new method will now be optimized for clinical use. Results from this research have been published in EMBO Molecular Medicine.

Epilepsy affects approximately 5 million people in Europe. The disorder is characterized by the recurrent and synchronized firing of groups of nerve cells. The propagation of these electrical discharges interrupts normal brain function and produces an epileptic seizure. The most common form of epilepsy is known as temporal lobe epilepsy (TLE), which is characterized by seizures originating in the temporal lobes. Long-term consequences of TLE can include memory problems as well as impaired learning and emotional control. Additionally, TLE patients’ quality of life is severely affected by restrictions on their ability to work, drive a car or do sports. This is further compounded by the fact that the drugs used to treat TLE patients are often unable to adequately control the disorder yet are still associated with severe side effects. For this particular group of patients, surgical removal of the temporal lobe frequently remains the only treatment alternative. However, this treatment is associated with adverse cognitive outcomes and does not guarantee that patients will remain free from seizures. Working alongside Prof. Dr. Christoph Schwarzer (Medical University of Innsbruck’s Department of Pharmacology), Prof. Dr. Regine Heilbronn (Director of the Institute of Virology on Charité’s Campus Benjamin Franklin) developed a new therapeutic concept for drug-resistant TLE.

The new treatment method is based on targeted gene therapy. This technique involves the selective delivery of a specific gene to nerve cells within the area of the brain from which the epileptic seizures originate. The gene provides the cells with the information needed to synthesize dynorphins. These are naturally produced peptides which modulate neural activity. Once the gene has been delivered into the nerve cells, it remains there permanently. The cells then start to produce and store dynorphins. Explaining the new technique’s mechanism of action, Prof. Schwarzer says: “High-frequency stimulation of the nerve cells, such as that seen at the beginning of a seizure, results in the release of stored dynorphins. Dynorphin dampens signal transduction and, as a result, the epileptic seizure doesn’t spread.” The neurobiologist and epilepsy expert goes on to add: “As the cells will only release this substance when needed, this type of gene therapy is referred to as ‘release-on-demand’.” Using an animal model, the researchers were able to show that this gene therapy is capable of suppressing epileptic seizures for several months. Reduction of seizures also meant relief from their adverse effects on memory and learning. Moreover, no side effects have been observed so far. This is most probably due to the site-specific release of dynorphin and its short duration of action. Thanks to the substance’s on-demand delivery, there was also no evidence of drug tolerance. The researchers then went on to test their new treatment concept using tissue samples obtained from epilepsy patients. They did so with great success, as dynorphin was shown to significantly reduce the severity and frequency of synchronized nerve cell activity in human epileptic tissue...

Published on EMBO Mol. Medicine (October 30, 2019):

https://doi.org/10.15252/emmm.201809963