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Researchers have discovered that the same cellular mechanism involved in a form of cystic fibrosis is also implicated in a form of a rare disease called cystinosis. The mechanism cleans up mutated proteins. In cystinosis, a genetic disease, this allows cystine crystals to build up in the cell. This disrupts the cell, and eventually, tissues and ultimately organs, particularly the kidneys and the eyes. University of Michigan researchers have discovered that the same cellular mechanism involved in a form of cystic fibrosis is also implicated in a form of a rare disease called cystinosis. The mechanism cleans up mutated proteins. In cystinosis, a genetic disease, this allows cystine crystals to build up in the cell. This disrupts the cell, and eventually, tissues and ultimately organs, particularly the kidneys and the eyes. The problem begins when the lysosome, an organelle within the cell, is unable to do its job. Often called the recycling center of the cell, the lysosome takes in cellular garbage, breaks it down into reusable cellular building blocks, then transports those materials back into the cell. But when the protein that transports one of the recycled amino acids back into the cell mutates and fails, a cellular mechanism cleans up the faulty protein, allowing amino acid, or cystine, to build up in the lysosome. "If cystinosis not treated at an early age, some of the effects are irreversible and it could include impaired growth, kidney failure and neurological problems," said Varsha Venkatarangan, graduate student in the U-M Department of Molecular, Cellular and Developmental Biology and lead author of the study. "Typically, the symptoms of the disease are treated rather than the root problem. So we have been wondering what could be the possible cellular mechanism of this disease." Venkatarangan worked with fibroblasts, skin tissue cells derived from patients with the disease. Using these cells, she determined that this disease mechanism called endoplasmic-reticulum-associated degradation, or ERAD, degrades a mutated version of the lysosome cystine transporter. ERAD is the same mechanism behind other diseases such as a major form of cystic fibrosis. And because this mechanism has been identified, the researchers were able to show that previously identified drug molecules were able to help the transporter protein remain stable. Their findings are now published in the Journal of Clinical Investigation. "This drug molecule is a small chemical that can somehow assist the protein folding of the mutant and protect it from being degraded," said MCDB professor Ming Li. "This chemical chaperone helps it achieve the right formation so that it can be functional." The lab, led by Li, became interested in the cystine transporting protein and wanted to understand how mutations in the transporter gene caused cystinosis. They painstakingly collected nearly 40 mutations that caused cystinosis and studied their protein stability. The researchers identified one striking mutation that had a very short half life compared to the non-mutated form of the protein. "We were interested about the fact that this disease mutant was degrading so rapidly, what was the potential mechanism by which it was degrading, and the machinery involved," Venkatarangan said. So Venkatarangan performed numerous genetic and chemical tests to inhibit different protein degradation machinery of the cell, narrowing down which cellular mechanism was involved in the disease. She determined that the mechanism was ERAD, a cellular pathway located in the cell's endoplasmic reticulum. The cell's endoplasmic reticulum is a network of tiny tubes and sheets that help ferry the cell's proteins to their destination. ERAD targets misfolded proteins and marks them for protein degradation. And because this is a commonly known disease mechanism, involved in cystic fibrosis, drug molecules had already been identified to work around the ERAD mechanism and prolong the life of the protein. These drug molecules, Li said, are called chemical chaperones and help the protein achieve the right formation so that they can be functional. "This small chemical chaperone can somehow magically -- we don't know the exact mechanism -- bind to our mutant and protect the protein from being degraded," Li said. The researchers were able to stabilize the protein and determine that it was in the right location where it could function to ferry amino acids out of the lysosome, but they then needed to determine whether the transporter was still functional. Performing an assay in collaboration with UCSD, the researchers measured the cystine concentration in the lysosome. With a functional transporter, the lysosome would have lower amounts of amino acid. "The reduction was pretty dramatic -- nearly a 70% reduction of the accumulation level," Li said. "That directly brought the system level to almost normal. If we actually used this chemical chaperone to treat patients, it's possible that we could directly bring down the cystine accumulation in the lysosome." People may wonder why a disease mechanism found in the endoplasmic reticulum might affect the function of lysosome membrane proteins, Li said. That's because lysosome membrane proteins are transported from the endoplasmic reticulum. While the lysosome is being built, it needs a lot of trafficking proteins. These lysosomal proteins go through the endoplasmic reticulum and then the Golgi apparatus, which packages the proteins and sends them to the lysosome. During protein trafficking, they have to fold properly. Otherwise, they will be degraded prematurely by protein quality control systems such as ERAD. These mutant proteins disappear early in the trafficking stage, the researchers discovered, after getting stuck and degraded in the endoplasmic reticulum. In finding a new purpose for a drug already in use, the team also fulfilled a goal of a program at the National Institutes of Health that encourages finding new uses for already-approved drugs as this leads to faster therapies. However, the team emphasizes that the work is preliminary, done only in cultured patient cells, and the drug has not been tested to treat cystinosis in animals or humans. "Again, this is preliminary work that was done in patient cells, so we don't really know the systemic effect or how well it works in actual patients," Venkatarangan said. "Our finding also emphasizes the importance of precision medicine, because most of the time, disease mutations are treated similarly under one umbrella of a particular disease. "But the fact that individual mutations may actually have their own pathogenesis mechanism emphasizes that understanding the individual patient mechanism might help them with a better treatment strategy." Co-authors of the paper include U-M MCDB's Weichao Zhang and Xi Yang. Jess Thoene of the Department of Pediatrics at the U-M Medical School and Si Houn Hahn of the Seattle Children's Hospital at University of Washington School of Medicine also contributed to the study.

All patients remain off oral cysteamine up to 36 months post gene therapy Sustained engraftment and durable reduction in leukocyte cystine levels across all patients Received positive regulatory feedback from US and UK agencies CAMBRIDGE, Mass.--(BUSINESS WIRE)-- AVROBIO, Inc. (Nasdaq: AVRO), a leading clinical-stage gene therapy company working to free people from a lifetime of genetic disease, today announced follow-up data demonstrating a durable treatment effect across key measures out to 36 months from a collaborator-sponsored Phase 1/2 clinical trial1 evaluating an investigational gene therapy for the treatment of cystinosis. These data are being presented at the 26th Annual Meeting of the American Society of Gene and Cell Therapy (ASGCT) in Los Angeles, California, on May 18, 2023. Cystinosis is a rare, progressive disease with a high treatment burden and unmet need. The fully enrolled Phase 1/2 clinical trial is monitoring long-term safety and efficacy in six adult patients affected by the most severe and common form of cystinosis who previously had been treated with the standard of care (SOC), cysteamine. The patients’ own hematopoietic stem cells (HSCs) were genetically modified to express a functional version of cystinosin, the protein that is deficient in people living with cystinosis. Preliminary data suggest that post-gene therapy, functional cystinosin is produced throughout the body as evidenced by clinical measures in multiple tissues, including the eyes, skin and gastrointestinal mucosa, as well as by neurocognitive tests suggestive of activity in the central nervous system. No adverse events (AEs) related to the drug product or serious adverse events have been reported to date. “These data show that genetically modifying a patient’s own HSCs has the potential to restore functional cystinosin and systemically reduce the accumulation of cystine, laying the foundation for a registration-enabling clinical trial,” said AVROBIO Chief Medical Officer Essra Ridha, M.D., MRCP, FFPM. “We are excited about moving this investigational gene therapy closer to patients.” In addition to the data presented, the Company also announced positive and productive meetings with the U.K. Medicines and Healthcare products Regulatory Agency (MHRA) and U.S. Food and Drug Administration (FDA) designed to align on regulatory paths and obtain feedback on this program. Data show investigational HSC gene therapy durably and systemically impacts neurocognitive measures and reduces cystine levels in the blood, and crystal accumulation in the skin and gastrointestinal mucosa Follow-up data suggest that after receiving HSC gene therapy, patients can produce functional cystinosin protein throughout the body. As a result, leukocyte cystine levels in the blood have decreased below baseline in all six patients, and stabilized up to 36 months out from treatment. Skin and gastrointestinal mucosa biopsies reveal a decline in tissue cystine crystals below baseline in the first four patients, who have been observed for at least 12 months, with two patients observed up to 24 months. Patients with cystinosis typically do not see an improvement in visual spatial or visual motor function over time in standardized tests evaluating the ability of the brain to interpret and translate visual information into an exact motor response. The first four patients treated with gene therapy have shown an improvement or stabilization of scores on the Beery – Buktenica Developmental Test of visual motor integration, up to 36 months out, suggesting a potential impact on the neuropathology of the disease. These data represent an extension of trends that have previously been measured, confirming the durability of the treatment effect up to 36 months. Safety and tolerability pro strong Preliminary data from this trial suggest that this HSC gene therapy is well tolerated, with no AEs related to the drug product to date. All AEs were related to myeloablative conditioning, stem cell mobilization, the underlying disease, co-morbid or pre-existing conditions. The majority of AEs were mild or moderate and resolved without clinical sequelae. An oral presentation by Dr. Cherqui on these data, “Phase 1/2 Clinical Trial of Autologous Hematopoietic Stem and Progenitor Cell Gene Therapy for Cystinosis,” will occur today at 3:45 PM PT in the session Metabolic, Storage, Endocrine, Liver and Gastrointestinal Disease II of the ASGCT Annual Meeting. Further details on the Phase 1/2 trial (NCT03897361) are available on clinicaltrials.gov. About cystinosis Cystinosis is a rare, progressive disease marked by the accumulation of cystine in cellular organelles known as lysosomes. This buildup causes progressive organ damage and debilitating corneal damage, swallowing dysfunction, chronic kidney disease leading to end-stage renal disease and muscle wasting leading to a shortened lifespan. Currently, more than 90% of treated cystinosis patients require a renal transplant in the second or third decade of life. The current standard of care for cystinosis is cysteamine, a treatment regimen that can require dozens of pills per day, does not prevent overall disease progression and carries side effects, such as breath and body odor and gastrointestinal complications, which may be difficult to tolerate. About AVROBIO Our vision is to bring personalized gene therapy to the world. We target the root cause of genetic disease by introducing a functional copy of the affected gene into patients’ own hematopoietic stem cells (HSCs), with the goal of durably expressing the therapeutic protein throughout the body, including the central nervous system. Our first-in-class pipeline includes clinical programs for Gaucher disease, cystinosis and Hunter syndrome, as well as a preclinical program for Pompe disease. Our proprietary plato® gene therapy platform is scalable for planned global commercialization. We are headquartered in Cambridge, Mass. For additional information, visit avrobio.com, and follow us on Twitter and LinkedIn. Forward-Looking Statements This press release contains forward-looking statements, including statements made pursuant to the safe harbor provisions of the Private Securities Litigation Reform Act of 1995. These statements may be identified by words and phrases such as “aims,” “anticipates,” “believes,” “could,” “designed to,” “estimates,” “expects,” “forecasts,” “goal,” “intends,” “may,” “plans,” “possible,” “potential,” “seeks,” “will,” and variations of these words and phrases or similar expressions that are intended to identify forward-looking statements. These forward-looking statements include, without limitation, statements regarding our business strategy for and the potential therapeutic benefits of our product candidates, including our AVR-RD-04 investigational gene therapy for the treatment of cystinosis, regulatory pathways, anticipated benefits of our gene therapy platform including potential impact on our commercialization activities, timing and likelihood of success, the expected benefits and results of our implementation of the plato platform in our clinical trials and gene therapy programs, and the expected safety pro our investigational gene therapies. Any such statements in this press release that are not statements of historical fact may be deemed to be forward-looking statements. Results in preclinical or early-stage clinical trials may not be indicative of results from later stage or larger scale clinical trials and do not ensure regulatory approval. You should not place undue reliance on these statements, or the scientific data presented. Any forward-looking statements in this press release are based on AVROBIO’s current expectations, estimates and projections about our industry as well as management’s current beliefs and expectations of future events only as of today and are subject to a number of risks and uncertainties that could cause actual results to differ materially and adversely from those set forth in or implied by such forward-looking statements. These risks and uncertainties include, but are not limited to, the risk that any one or more of AVROBIO’s product candidates, including AVR-RD-04 for the treatment of cystinosis, will not be successfully developed or commercialized, the risk of cessation or delay of any ongoing or planned clinical trials of AVROBIO or our collaborators, the risk that AVROBIO may not successfully recruit or enroll a sufficient number of patients for our clinical trials, the risk that AVROBIO may not realize the intended benefits of our gene therapy platform, including the features of our plato® platform, the risk that our product candidates or procedures in connection with the administration thereof will not have the safety or efficacy pro we anticipate, the risk that prior results, such as signals of safety, activity or durability of effect, observed from preclinical or clinical trials, will not be replicated or will not continue in ongoing or future studies or trials involving AVROBIO’s product candidates, the risk that we will be unable to obtain and maintain regulatory approval for our product candidates, the risk that the size and growth potential of the market for our product candidates will not materialize as expected, risks associated with our dependence on third-party suppliers and manufacturers, risks regarding the accuracy of our estimates of expenses and future revenue, risks relating to our capital requirements and needs for additional financing, risks relating to clinical trial and business interruptions resulting from the COVID-19 outbreak or similar public health crises, including that such interruptions may materially delay our enrollment and development timelines and/or increase our development costs or that data collection efforts may be impaired or otherwise impacted by such crises, and risks relating to our ability to obtain and maintain intellectual property protection for our product candidates. For a discussion of these and other risks and uncertainties, and other important factors, any of which could cause AVROBIO’s actual results to differ materially and adversely from those contained in the forward-looking statements, see the section entitled “Risk Factors” in AVROBIO’s most recent Quarterly Report, as well as discussions of potential risks, uncertainties and other important factors in AVROBIO’s subsequent filings with the Securities and Exchange Commission. AVROBIO explicitly disclaims any obligation to update any forward-looking statements except to the extent required by law. _____________________________ 1The collaborator-sponsored Phase 1/2 clinical trial for cystinosis is funded in part by grants to UCSD from the California Institute for Regenerative Medicine (CIRM), Cystinosis Research Foundation (CRF) and National Institutes of Health (NIH).

The rare genetic disease cystinosis is caused by mutations in the gene for a protein called cystinosin. A team of scientists has now solved the structure of cystinosin and determined how mutations interfere with its normal function, providing insights into the underlying mechanisms and suggesting a way to develop new treatments for the disease. The rare genetic disease cystinosis is caused by mutations in the gene for a protein called cystinosin. A team of scientists has now solved the structure of cystinosin and determined how mutations interfere with its normal function, providing insights into the underlying mechanisms and suggesting a way to develop new treatments for the disease. The new study, published September 15 in Cell, involved a collaborative effort by researchers at UC Santa Cruz, Stanford University, and the University of Texas Southwestern Medical Center, who combined their expertise in three specialized methods for studying protein structure and function: x-ray crystallography, cryogenic electron microscopy (cryo-EM), and double electron-electron resonance (DEER). "This paper could set a model for how to combine those three areas, along with biochemical assays, to quickly narrow in on how a protein functions and identify a therapeutic strategy," said Glenn Millhauser, distinguished professor and chair of chemistry and biochemistry at UC Santa Cruz and a corresponding author of the paper. Cystinosin is a specialized transporter protein that plays a crucial role in how cells manage the essential amino acid cysteine. Cells are constantly recycling proteins, breaking them down into their constituent amino acids for use in building new proteins. Transporters like cystinosin move the amino acids out of lysosomes -- the cellular compartments where proteins are broken down -- into the cell to be reused. When cystinosin isn't functioning properly due to mutations, a form of cysteine (a dimer called cystine) builds up inside the lysosomes. The abnormal accumulation of cystine causes widespread damage to tissues and organs and can lead to kidney failure, muscle wasting, and other problems. "It's a rare disease, but it can be deadly," Millhauser said. "If it's untreated, people with cystinosis usually die by age ten." Cystinosin adopts different conformations when it is open to the inside of the lysosome to load cystine and when it is open to the outside to release cystine. The research teams at Stanford (led by Professor Liang Feng) and at UT Southwestern (led by Professor Xiaochun Li) solved the structures of cystinosin in these different structural conformations using x-ray crystallography and cryo-EM. Understanding cystinosin's structural changes through the transport process, however, required the DEER studies performed by Millhauser's lab. DEER is a specialized magnetic resonance technique that can be used to determine how a protein changes its shape. "With that we were able to figure out the mechanism that allows cystinosin to switch between those different states, and we could narrow in on which of the protein's amino acids were driving the transition," Millhauser said. "Now we can see how the mutations are changing the protein's ability to change shape and pump cystine out of the lysosome." These new insights into the molecular mechanics of cystinosin's transport activity not only provide a more detailed understanding of the pathogenesis of cystinosis, but also suggest a possible therapeutic strategy to treat the disease. "It may be possible to enhance the transport activity of cystinosin by developing conformation-selective small molecules or biologics that favor a cytosol-open conformation," the authors wrote. A similar approach could be used to target other transporter proteins, which are involved in a wide range of diseases. The authors of the paper include co-first authors Tufa Assafa at UC Santa Cruz, Xue Guo at Stanford, and Philip Schmiege at UT Southwestern, and coauthors Yan Xu at Stanford, Rong Wang, Linda Donnelly, and Michael Fine at UT Southwestern, and Xiaodan Ni and Jiansen Jiang at the National Heart, Lung, and Blood Institute. This work was funded in part by the National Institutes of Health.