Virus World

The lone volunteer in a gene-editing study targeting a rare form of Duchenne muscular dystrophy likely died after having a reaction to the virus that delivered the therapy in his body, researchers concluded in an early study. The lone volunteer in a gene-editing study targeting a rare form of Duchenne muscular dystrophy likely died after having a reaction to the virus that delivered the therapy in his body, researchers concluded in an early study. Terry Horgan, 27, of Montour Falls, New York, died last year during one of the first tests of a gene-editing treatment designed for one person. Some scientists wondered if the gene-editing tool CRISPR played a part in his death. The tool has transformed genetic research, sparked the development of dozens of experimental drugs, and won its inventors the Nobel Prize in 2020. But researchers said the virus—one used to carry treatment into the body because it doesn't usually make people sick—combined with his condition, triggered the problems that ultimately killed him. Horgan appears to have had a more severe immune reaction "than others receiving similar or slightly higher doses" of the virus, the authors wrote in the study, which has not yet been peer-reviewed. Horgan was enrolled in an early-stage safety trial approved by the Food and Drug Administration. It was sponsored by Cure Rare Disease, a Connecticut-based nonprofit founded by his brother, Rich, to try and save him from the muscle-wasting disease caused by a mutation in the gene needed to produce a protein called dystrophin.

In a statement, Rich Horgan thanked the research team led by the University of Massachusetts Chan Medical School and Yale University for a "thorough, comprehensive" investigation that provided valuable insights. He added, "On a personal note, this study is another important step toward honoring Terry's legacy and his commitment, as well as our entire family's, to the rare disease community." The therapy Horgan got aimed to use CRISPR to increase a form of the dystrophin protein. The process began with suppressing Horgan's immune system to prepare his body for the therapy, which was delivered by IV with "a high dose" of what's known as an adeno-associated viral vector, or AAV, according to Cure Rare Disease. But Horgan soon began experiencing problems, went into cardiac arrest six days after the treatment and died two days later from organ failure and brain damage. Because of the timing of symptoms, and the fact researchers could find little of a gene-editing enzyme in his body, they concluded that the therapy hadn't been activated yet. This isn't the first time viral vectors have been implicated in a gene therapy trial death. In a major setback for the field, 18-year-old Jesse Gelsinger died in 1999 during a study aimed at combatting his rare metabolic disease. Scientists later learned that his immune system overreacted to the virus used to carry the treatment. The virus used in Horgan's trial is considered safer but it is not without problems. "People have been trying to make safer vectors … but they still remain challenging," said Arthur Caplan, a medical ethicist at New York University who was not involved in the study but has followed the case closely. "We don't really understand why some people run into trouble and others don't. We don't know whether it's their underlying disease, some co-morbidity, or some strange immunology." Rich Horgan said they plan to submit the study to a peer-reviewed journal. Meanwhile, Cure Rare Disease said it will use alternative viruses for the other treatments it is trying to develop. Dr. Terence Flotte, dean of the UMass medical school and senior author of the study, said he hopes it leads "to further research into how to identify subsets of patients who might be prone to severe, unexpected reactions like this."

Cited research available in  medRxiv (May 30, 2023):

 https://doi.org/10.1101/2023.05.16.23289881 

Infection with multiple common viruses may be responsible for the cases that puzzled doctors last year. Last year, reports of severe, unexplained hepatitis in previously healthy children puzzled health experts around the world. Now, a small new study of American children adds to the evidence that the cases, which remained extremely rare, may have been caused by a simultaneous infection with multiple common viruses, including one known as adeno-associated virus type 2, or AAV2. AAV2 is not typically associated with disease, and it requires a second “helper” virus in order to replicate. Many of the children with unexplained hepatitis, or liver inflammation, were infected with multiple helper viruses, the researchers found. Although the idea remains speculative, the timing of the outbreak may have been related to the loosening of pandemic precautions, leaving large numbers of young children exposed to common viruses they had not previously encountered. “It may have resulted in a population that was highly vulnerable to getting infected with multiple viral infections,” said Dr. Charles Chiu, an infectious disease specialist and microbiologist at the University of California, San Francisco, and an author of the new study. The research appeared Thursday in the journal Nature, alongside two British studies that also implicated AAV2 in the hepatitis cases. Preliminary versions of the British studies were posted online last summer. The consistent findings are “quite striking,” said Dr. Frank Tacke, head of the gastroenterology and hepatology department at the Charité University Medical Center in Berlin, who was not involved in the research but wrote an accompanying commentary. “The fact that three independent groups found this from different areas of the world actually makes it really convincing.” Still, the findings are not definitive, and many uncertainties remain, including how these infections might trigger hepatitis and whether AAV2 plays a causal role or is “just a bystander,” Dr. Tacke said. (There has also been some debate about whether the cases truly became more commonlast year or whether they were part of a previously unrecognized phenomenon.) The cases date back to the fall of 2021 but seemed to peak last spring and summer before tapering off, experts said. By last July, more than 1,000 probable cases had been reported in 35 countries, including the United States, according to the World Health Organization. Roughly 5 percent of children required liver transplants, and 2 percent died. In several early studies, scientists found that many of the affected children were infected with adenoviruses, particularly adenovirus 41, which typically causes gastrointestinal symptoms. Adenoviruses are not typically known to cause hepatitis in otherwise healthy children, but they are common helper viruses for AAV2.

Researchers have identified a novel cellular entry factor for adeno-associated virus vector (AAV) types -- the most commonly used viral vectors for in vivo gene therapy. The researchers identified that GPR108, a G protein-coupled receptor, served as a molecular 'lock' to the cell. The discovery could one day enable scientists to better direct AAV gene transfers to specific tissues. The researchers identified that GPR108, a G protein-coupled receptor, served as a molecular 'lock' to the cell. GPR108 is required for most AAVs, including those used in approved gene therapies, to gain access to the cell. As gaining cellular access is a critical step in delivering gene therapy, this discovery may provide a crucial piece of information that could one day enable scientists to better explain, predict, and ultimately, direct AAV gene transfers to specific tissues. The study was recently published in Molecular Therapy.

"For years we have known that AAV gene transfer is highly effective, but we have yet to learn how that is achieved and why some AAV types function differently than others," said senior study author Luk Vandenberghe, PhD, Director of the Grousbeck Gene Therapy Center at Mass. Eye and Ear and Associate Professor of Ophthalmology at Harvard Medical School. "We identified a molecular 'lock' to the cell that allows AAV vectors carrying the appropriate 'key' to gain access to the cell. This finding may enable scientists to better direct AAV gene transfers to targeted cell tissues, in order to treat specific genetic diseases."

Multiple AAV types are in clinical trials for diseases affecting the eye, muscles, and neurons. Luxturna™ and Zolgensma™, both recently approved by the U.S. Food and Drug Administration, are AAV gene therapy products for a form of blindness and neuromuscular disease. Yet, the exact mechanism by which this novel class of medicine accomplishes gene transfer has remained poorly understood.

Published in Molecular Therapy (November 13, 2019):

https://doi.org/10.1016/j.ymthe.2019.11.005

Novel spinal therapy/delivery approach prevented disease onset in neurodegenerative ALS disease model in adult mice and blocked progression in animals already showing disease symptoms. Writing in Nature Medicine, an international team headed by researchers at University of California San Diego School of Medicine describe a new way to effectively deliver a gene-silencing vector to adult amyotrophic lateral sclerosis (ALS) mice, resulting in long-term suppression of the degenerative motor neuron disorder if treatment vector is delivered prior to disease onset, and blockage of disease progression in adult animals if treatment is initiated when symptoms have already appeared.

Writing in Nature Medicine, an international team headed by researchers at University of California San Diego School of Medicine describe a new way to effectively deliver a gene-silencing vector to adult amyotrophic lateral sclerosis (ALS) mice, resulting in long-term suppression of the degenerative motor neuron disorder if treatment vector is delivered prior to disease onset, and blockage of disease progression in adult animals if treatment is initiated when symptoms have already appeared. The findings are published in the December 23, 2019 online issue of the journal Nature Medicine. Martin Marsala, MD, professor in the Department of Anesthesiology at UC San Diego School of Medicine and a member of the Sanford Consortium for Regenerative Medicine, is senior author of the study. 

ALS is a neurodegenerative disease that affects nerve cells in the brain and spinal cord. Motor neurons responsible for communicating movement are specifically harmed, with subsequent, progressive loss of muscle control affecting the ability to speak, eat, move and breathe. More than 5,000 Americans are diagnosed with ALS each year, with an estimated 30,000 persons currently living with the disease. While there are symptomatic treatments for ALS, there is currently no cure. The majority of patients succumb to the disease two to five years after diagnosis. There are two types of ALS, sporadic and familial. Sporadic is the most common form, accounting for 90 to 95 percent of all cases. It may affect anyone. Familial ALS accounts for 5 to 10 percent of all cases in the United States, and is inherited. Previous studies show that at least 200 mutations of a gene called SOD1 are linked to ALS. The SOD1 gene normally serves to provide instructions for making an enzyme called superoxide dismutase, which is widely used to break down superoxide radicals — toxic oxygen molecules produced as a byproduct of normal cell processes. Previous research has suggested that SOD1 gene mutations may result in ineffective removal of superoxide radicals or create other toxicities that cause motor neuron cell death, resulting in ALS.

The new approach involves injecting shRNA — an artificial RNA molecule capable of silencing or turning off a targeted gene — that is delivered to cells via a harmless adeno-associated virus. In the new research, single injections of the shRNA-carrying virus were placed at two sites in the spinal cord of adult mice expressing an ALS-causing mutation of the SOD1 gene, either just before disease onset or when the animals had begun showing symptoms....

Published in Nature Medicine (23 Dec. 2019):

https://doi.org/10.1038/s41591-019-0674-1

Neuroscientists at Lund University in Sweden have developed a new technology that engineers the shell of a virus to deliver gene therapy to the exact cell type in the body that needs to be treated. The researchers believe that the new technology can be likened to dramatically accelerating evolution from millions of years to weeks. Several of the new revolutionary treatments that have been used clinically in recent years to treat complex diseases—such as spinal muscular atrophy and enzyme deficiency—are based on gene therapy. With gene therapy, the genetic material is controlled or altered using biological drugs. Examples of this are the gene scissors CRISPR / Cas9 and the so-called CAR-T cells that are used to treat various forms of cancer. This type of treatment is often engineered by growing viruses in the laboratory. The viruses are altered so that they are harmless and can deliver new genetic material to the body's cells, replacing the damaged genome. The virus's own genome, which is required for it to spread, has been completely removed.

In the last five years, neuroscientist Tomas Björklund and his research group have developed a process that tailors these virus shells, or virus capsids, so that they can reach precisely the cell type in the body that needs to be treated, for example nerve cells. The process combines powerful computer simulations and modeling with the latest gene technology and sequencing technology. "Thanks to this technology, we can study millions of new virus variants in cell culture and animal models simultaneously. From this, we can subsequently create a computer simulation that constructs the most suitable virus shell for the chosen application– in this case, the dopamine-producing nerve cells for the treatment of Parkinson's disease," says Tomas Björklund, senior lecturer in translational neuroscience at Lund University. "You can view this as dramatically speeding up evolution from millions of years to weeks. The reason we can do this is that we study each "generation" of the virus in parallel with all the others in the same nerve cells. Unlike evolution, where only the best suited live on to the next generation, we can also learn what makes the virus work less well through this process. This is crucial when building computer models that interpret all the information," he continues. 

With the new method, researchers have been able to significantly reduce the need for laboratory animals, as millions of variants of the same drug are studied in the same individual. They have also been able to move important parts of the study from animals to cell culture of human stem cells. "We believe that the new synthetic virus we succeeded in creating would be very well suited for gene therapy for Parkinson's disease, for example, and we have high hopes that these virus vectors will be able to be put into clinical use. Together with researchers at Harvard University, we have established a new biotechnology company in Boston, Dyno Therapeutics, to further develop the virus  engineering technology, using artificial intelligence, for future treatments," concludes Tomas Björklund.

Published in P.N.A.S. (December 9, 2019):

https://doi.org/10.1073/pnas.1910061116

The US Food and Drug Administration halts a study by Solid Biosciences after a patient experiences severe side effects following treatment. A clinical trial of a gene therapy for Duchenne muscular dystrophy has been halted after a patient suffered serious side effects following treatment, Reuters reports today (November 12). After receiving Solid Biosciences’s experimental therapy, SGT-001, the patient experienced kidney injury and drops in red blood cell count, leading the US Food and Drug Administration (FDA) to place the study on hold.

“We are encouraged that this patient is recovering,” Ilan Ganot, Solid Biosciences’s CEO, president, and cofounder, says in a statement. “In the coming weeks, we anticipate that we will have a better understanding of the biological activity and potential benefit of SGT-001. We look forward to sharing this additional data and working with the FDA to resolve the clinical hold and determining next steps for the program.” SGT-001 has been administered to six people so far, and involves the transfer of an engineered version of the dystrophin gene DMD, which is dysfunctional in people with Duchenne muscular dystrophy, using an adeno-associated virus (AAV) as a vector. Sarepta Therapeutics, Pfizer, and other biopharmaceutical companies are investigating similar approaches to treat the condition, although the choice of AAV varies.

This isn’t the first time Solid Biosciences’s trial of SGT-001 has been put on hold. Early last year, the FDA halted the same study after a patient receiving a low dose of the therapy experienced a drop in red blood cell count and had to be hospitalized. The company was allowed to resume the trial last June after making changes to the study design. In February, the company reported disappointing interim results from the first stage of the trial, which administered a low dose of the therapy to the first three patients in the study. The patient mentioned in today’s announcement was one of three patients subsequently given a higher dose. Shares in the company dropped 71 percent following today’s announcement, according to Reuters.

The FDA has imposed a partial hold on clinical trials for intrathecal administration of the Novartis gene therapy AVXS-101, which won the FDA's first approval for treating some forms of spinal muscular atrophy (SMA) in May under the name Zolgensma® (onasemnogene abeparvovec-xioi). The partial hold does not affect the marketing of Zolgensma or clinical trials assessing intravenous (IV) delivery of AVXS-101, Novartis emphasized. However, the hold affects studies assessing AVXS-101 administered as an injection into the spinal canal in patients with SMA Type 2.

As a result of the hold, enrollment in the high dose cohort has been stopped in the Phase I STRONG trial (NCT03381729), an ongoing, open-label, dose-comparison, multi-center trial designed to evaluate the efficacy, safety, and tolerability of one-time intrathecal administration of AVXS-101. The low- and mid-dose cohort enrollment has previously been completed and interim results have been presented. The pharma giant said the partial hold followed AveXis, A Novartis Company, alerting authorities and clinical trial investigators about animal findings from a small, AveXis-launched preclinical study showing dorsal root ganglia (DRG) mononuclear cell inflammation, sometimes accompanied by neuronal cell body degeneration or loss.

“The clinical significance of the DRG inflammation observed in this preclinical animal study is not known and was not seen in prior animal studies with AVXS-101,” Novartis said in a statement, adding that DRG inflammation can be associated with sensory effects. “We have completed a thorough review of human safety data from all available sources to date and no adverse effects related to sensory changes have been seen in AVXS-101 intrathecal or Zolgensma. We are working with health authorities to confirm further guidance to clinical investigators.”  Novartis also said it will continue to closely monitor for any reports of related safety events in patients, adding: “We remain confident that the overall benefit-risk profile for patients on treatment is favorable.”

The partial clinical hold comes a month after another safety issue related to the gene therapy. Last month, Novartis acknowledged the death of a six-month-old patient treated with Zolgensma in the European Phase III clinical trial STRIVE-EU (NCT03461289)—but insisted that the death was not the result of toxicity within the gene therapy. “According to the coroner’s report, the immediate cause of death was hypoxic-ischemic brain damage with respiratory tract infection as the underlying cause,” AveXis stated on September 19. “SMA Type 1 was indicated as the underlying cause for the respiratory tract infection. In addition, there was no evidence of an inflammatory CNS process or a toxic or a treatment-related brain damage.” Zolgensma is an adeno-associated virus vector-based gene therapy that won FDA approval on May 24. Zolgensma is indicated for the treatment of SMA in pediatric patients less than two years of age with SMA with bi-allelic mutations in the survival motor neuron 1 (SMN1) gene.....

A gene therapy being developed at Penn Medicine to treat Duchenne muscular dystrophy (DMD) successfully and safely stopped the severe muscle deterioration associated with the rare, genetic disease in both small and large animal models, according to a first-of-its-kind study from Penn Medicine researchers. The findings, published online today in Nature Medicine, puts the field within closer reach of a safe and effective gene therapy that uses a "substitute" protein without triggering immune responses known to hinder other therapeutic approaches.

Found mostly in boys, DMD is caused by mutations in a sex-linked gene that stop production of a muscle-building protein known as dystrophin. Without it, muscles progressively deteriorate and weaken starting at a very young age and only worsen from there. Most patients aren't able to walk by age 12 and die of heart or respiratory failure by the time they reach their 30s, though respirators have helped some live longer. With their modified gene therapy approach, a multidisciplinary team from the Perelman School of Medicine at the University of Pennsylvania engineered adeno-associated virus (AAV) vectors to deliver a "substitute" protein for dystrophin in small and large animal DMD models to keep the muscles intact. The synthetic substitute, based on a naturally occurring protein called utrophin, proved to be an effective and safe alternative, as it protected muscle in mice and dogs with naturally occurring DMD-like mutations, including a large deletion that closely mirrors the large dystrophin deletions found in humans.

"For the first time, we've shown how a carefully constructed version of a dystrophin-related protein can safely prevent the breakdown of muscle and maintain its function over time in the most informative animal models. This discovery has important implications for gene therapy and how we work toward safe and effective treatments for muscular dystrophy," said senior author Hansell H. Stedman, MD, an associate professor of Surgery. "With these results, we have a strong rationale to move this forward into human clinical trials."

Restoring levels of dystrophin with gene therapy and other techniques have been under scrutiny due to the immune system's adverse reaction to additions it deems foreign. DMD patients have little to none of this protein, so their bodies can attack well-intentioned direct replacement proteins because they are seen as foreign. However, because dystrophin's distant cousin utrophin is expressed in other places in the body, it was thought that the immune system wouldn't view it as a threat. The Penn team showed this to be true in rigorous, randomized mouse and dog studies. Delivering a single-dose treatment of synthetic utrophin with the AAV vector to newborn mice showed distribution of the protein throughout the body, no signs of toxicity, and complete suppression of all signs of DMD, compared to untreated mice. The mice also sustained expression of utrophin in skeletal and cardiac muscles throughout that time, and physical tests in the mice supported the sustained muscle function.....

Published in Nature Medicine on October 7, 2019:

https://doi.org/10.1038/s41591-019-0594-0

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 

A five-year-old girl with Mucopolysaccharidosis Type IIIA (MPS IIIA) has died in the Phase II/III AAVance clinical trial (NCT03612869), designed to evaluate the efficacy of Lysogene’s gene therapy candidate LYS-SAF302 in improving or stabilizing the neurodevelopmental state of MPS IIIA patients, the company has acknowledged. In a statement, Lysogene said the immediate cause of death was unknown, that additional information was being collected, and that no evidence had surfaced linking the death to the administration of LYS-SAF302. “Lysogene is profoundly saddened by the passing of this child and extends its deepest sympathies to the family. The company remains committed to the LYS-SAF302 development program and the Sanfilippo patient community,” the company said October 15 in a statement.  The company added that per study protocol, it was “diligently” following the 18 other patients who have been treated in the AAVance trial, conducted at eight clinical sites. The girl died at home several months after receiving the therapy, which Lysogene told French news agency AFP consisted of a single injection administered at one of the four U.S. clinical sites. Lysogene shares have tumbled 21% since news of the death became public, falling to €1.99 ($2.36) at the close of trading Friday, from €2.53 ($3.00) the day before the announcement.

Earlier clinical hold

The FDA on June 5 issued a clinical hold for the trial following observations in some patients of localized findings on MRI images at the intracerebral injection sites, suggesting a potential connection to delivery, Lysogene disclosed. But the company added that “no clinical symptoms have been observed that could be directly attributed to the observed MRI findings.” LYS-SAF302 is an adeno-associated virus (AAV)-mediated gene therapy for MPS IIIA, also called Sanfilippo syndrome type A, that is designed to work by replacing the faulty SGSH (N-sulfoglucosamine sulfohydrolase) gene with a healthy copy of the gene. SGSH is involved in producing an enzyme necessary for the breakdown and disposal of long chain sugar molecules. MPS III is caused by mutations in the SGSH gene, which encodes Heparan-N-sulfamidase for heparan sulfate (HS) recycling in cells. LAF-SAF302 is intended to deliver a functional copy of the SGSH gene and allow the brain to secrete the missing enzyme. LYS-SAF302 employs the AAVrh10 virus, chosen for its ability to target the CNS. The trial is designed to show improved or stabilized neurodevelopmental status of MPS IIIA patients.

Trial to enroll up to 20 patients

LAF-SAF302 is intended to deliver a functional copy of the SGSH gene and allow the brain to secrete the missing enzyme. The goal of the trial is to show improved or stabilized neurodevelopmental status of MPS IIIA patients. The trial was designed to enroll up to 20 patients at eight sites in the United States and Europe. Lysogene granted Sarepta Therapeutics exclusive commercial rights to LYS-SAF302 in the United States and other markets outside Europe in 2018. Sarepta agreed to pay Lysogene $15 million at the close of their licensing agreement, up to approximately $125 million in development, regulatory, and commercial milestone payments, plus sales-based royalties. Sarepta also made an equity investment in Lysogene of $2.5 million under the licensing agreement, which included Lysogene granting to Sarepta option rights to an additional CNS-targeted gene therapy candidate. Lysogene’s patient death occurred months after the deaths of three patients in the Phase I/II ASPIRO trial (NCT03199469), in which Audentes Therapeutics, an Astellas company, was evaluating its gene therapy candidate AT132 in patients with X-linked Myotubular Myopathy (XLMTM). All three were treated with the trial’s high dose of 3×1014 GC/kg, and began to demonstrate signs of liver dysfunction within 3 to 4 weeks after dosing. The AT132 program remained on clinical hold as of October 16.

Just as gene therapy finally seems to be living up to its promise, a study has revived a lingering worry about the viral vessel that many efforts rely on to ferry therapeutic genes into patients. This “vector,” a stripped-down version of adeno-associated virus (AAV), was thought to be safe because it rarely knits the human DNA it carries into a cell’s chromosomes, where it might activate cancer-causing genes. But a study of dogs with hemophilia, treated with AAV up to 10 years earlier, has shown that the vector can readily insert its payload into the host’s DNA near genes that control cell growth. The new data, described in a conference talk last month by a Philadelphia-based research team, are “good news and bad news,” says gene therapy researcher Charles Venditti of the National Human Genome Research Institute. By slipping into the chromosomes rather than floating free, the therapeutic DNA might have longer lasting benefits. Integration “happens and it may actually be essential for long-term expression” of a needed protein, says physician-scientist David Lillicrap of Queen’s University, who attended the 9 December 2019 talk at the annual meeting of the American Society of Hematology (ASH) in Orlando, Florida. But the findings also fuel a debate about whether AAV vectors could pose an unacceptable cancer risk. “We don’t know enough yet” to say, according to Lillicrap.

Another viral vector, used in some early gene therapy trials, caused cancer in a few children after it integrated its cargo into the chromosomes. AAV seemed to be a safer alternative because genes introduced by the modified virus generally become a free-floating loop, known as an episome, in the cell’s nucleus. AAV vectors have helped drive the recent surge of successful gene therapies. These include one approved by the U.S. Food and Drug Administration last year for spinal muscular atrophy, a fatal childhood neurological disease, and a treatment for the blood-clotting disorder hemophilia B that’s expected to receive FDA approval this year. In the hemophilia treatment, AAV infects liver cells and turns the organ into a factory for making the clotting protein that patients depend on. Yet doubts about AAV’s safety have simmered for nearly 20 years, since a study found that in newborn mice given high doses of the virus, it could integrate its genetic cargo into the animals’ DNA and cause liver cancer. Many gene therapists argued that findings in newborn mice are not relevant to human adults.

But the new warning comes from larger, older animals: adult dogs with hemophilia A, in which a clotting protein called factor VIII is missing. In seven of nine such dogs, AAV vectors successfully supplied a replacement copy of the gene for factor VIII and restored stable production of the molecule. In two of those dogs, however, blood levels rose further after about 3 years, reaching about four times the original level by 7 to 8 years, gene therapy researcher Denise Sabatino of the Children’s Hospital of Philadelphia reported at the ASH meeting. After ending the experiment and studying the livers of six dogs, her team found that in every one of them, AAV-ferried DNA—the factor VIII gene or, more often, fragments of regulatory sequences—had integrated in many spots across the genome in the dogs’ liver cells, sometimes near genes affecting cell growth. Some of those cells had divided more than other cells, forming pockets of multiple cells or “clones” in some animals. Sabatino’s team suspects—although it can’t prove—that the insertions activated growth genes, explaining both the clones and the rise in blood levels of factor VIII in the two dogs......

Published in Science (Jan 6, 2019):

https://doi.org/10.1126/science.aba7696

Ring Therapeutics, a Flagship Pioneering spinout, launched Thursday with ambitious plans to expand the universe of vectors available for gene therapy. Gene therapy, treatments intended to treat disease by inserting a gene instead of using drugs or surgery, has had a banner year, with the second ever such therapy approved this year in the US. Ring want to use its research into viruses that exist in the human body without apparent negative effects to provide more and better options to fuel the rise of gene therapy treatments. For the past two years, Flagship Pioneering partner and Ring’s founding CEO Avak Kahvejian says the company has been exploring the human commensal virome—basically, a group of viruses that exist within humans without negative effects—for its potential to address limitations of the vectors currently used.

The sector relies heavily on adeno-associated viruses (AAVs), which naturally infect humans but aren’t known to cause disease, to deliver the DNA. Previous exposure, however, can spark an immune response. “A lot of the workhouses in gene therapy have either been pathogenic viruses or viruses that have been taken from other species or viruses that are highly immunogenic, or all of the above,”  Kahvejian tells Xconomy. “That leads to a certain number of limitations, despite the successes and advances we’ve made to date.” A number of issues stymie widespread use of AAVs, Kahvejian says, including the fact that 10 percent to 20 percent of people have at one time or another been infected with such a virus, thereby building up an immune response to it. Another concern is where such gene therapies end up, because viruses tend to gravitate toward certain types of tissues, and to go elsewhere, require special tweaking.

The Cambridge, MA-based startup believes the viruses it has found are unlikely to cause an immune response or prove pathogenic, given their ubiquity in the body. Like extrachromosomal DNA—a new discovery at least one company is exploring for its potential as a target in cancer treatments—the viral sequencing Ring is studying are circular pieces of DNA that exist outside the 23 chromosomes of the human genome. Ring says it has found thousands of these viruses that coexist with our immune system. It aims to use those to develop vectors that can facilitate gene replacement throughout the body—multiple times, if necessary. While gene therapy is thought of as a one-time fix, cell turnover means whatever the “fix” engendered by the inserted gene could falter over time, necessitating a re-up.

Press Release (Dec. 19, 2019)  available here:

https://www.prnewswire.com/news-releases/flagship-unveils-newest-pioneering-platform-ring-therapeutics-300976832.html

Dyno Therapeutics announces a publication in Science demonstrating the power of a comprehensive machine-guided approach to improve adeno-associated virus (AAV) capsids for gene therapies. Working to surpass the few naturally occurring AAV capsids currently in use, the authors synthesized large libraries of capsids and discovered changes that improve key properties preventing current AAVs from optimal therapeutic function. Dyno is a biotechnology company pioneering the use of artificial intelligence in gene therapy.

AAV capsids are presently the most commonly used vector for gene therapy because of their established ability to deliver genetic material to patient organs with a proven safety profile. However, there are only a few naturally occurring AAV capsids, and they are deficient in essential properties for optimal gene therapy, such as targeted delivery, evasion of the immune system, higher levels of viral production, and greater transduction efficiency. Starting at Harvard in 2015, the authors set out to overcome the limitations of current capsids by developing new machine-guided technologies to rapidly and systematically engineer a suite of new, improved capsids for widespread therapeutic use.

In the research published in Science, the authors demonstrate the advance of their unique machine-guided approach to AAV engineering. Previous approaches have been limited by the difficulty of altering a complex capsid protein without breaking its function and by the general lack of knowledge regarding how AAV capsids interact with the body. Historically, rather than addressing this challenge directly, the most popular approaches to capsid engineering have taken a roundabout solution: generating libraries of new capsids by making random changes to the protein. However, since most random changes to the capsid actually result in decreased function, such random libraries contain few viable capsids, much less improved ones. Recognizing the limitation of conventionally generated capsid libraries, the authors implemented a machine-guided approach that gathered a vast amount of data using new high-throughput measurement technologies to teach them how to build better libraries and, ultimately, lead to synthetic capsids with optimized delivery properties. Focusing on the AAV2 capsid, the authors generated a complete landscape of all single codon substitutions, insertions and deletions, then measured the functional properties important for in vivo delivery. They then used a machine-guided approach, leveraging these data to efficiently generate diverse libraries of AAV capsids with multiple changes that targeted the mouse liver and that outperformed AAVs generated by conventional random mutagenesis approaches. In the process, the authors' systematic efforts unexpectedly revealed the existence of a previously-unrecognized protein encoded within the sequence of all the most popular AAV capsids, which they termed membrane-associated accessory protein (MAAP). The authors believe that the protein plays a role in the natural life cycle of AAV.

"This is just the beginning of machine-guided engineering of AAV capsids to transform gene therapy," underscores co-author Sam Sinai, Ph.D., Lead Machine Learning Scientist and co-founder of Dyno Therapeutics. "The success of the simple linear models used in this study has led us to pursue more data and higher capacity machine learning models, where the potential for improvement in capsid designs feels boundless." 

"The results in the Science publication demonstrate, for the first time, the power of linking a comprehensive set of advanced techniques - large scale DNA synthesis, pooled in vitro and in vivo screens, next-generation sequencing readouts, and iterative machine-guided capsid design - to generate optimized synthetic AAV capsids," explains co-first and co-corresponding author Eric D. Kelsic, Ph.D., CEO and co-founder of Dyno Therapeutics. "At Dyno, our team is committed to advancing these technologies to identify capsids that meet the urgent needs of patients who can benefit from gene therapies."....

Published in Science (29 November, 2019):

https://doi.org/10.1126/science.aaw2900

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

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

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