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Home > Press > CRISPR/Cas9 therapeutic for tyrosinemai type I delivered to mice: AAV and lipid nanoparticles used to deliver CRISPR/Cas9 formulation capable of repairing genetic disease in animal models Abstract: University of Massachusetts Medical School researchers have found a way to more efficiently delivery a CRISPR/Cas9 therapeutic to adult mice with the metabolic disease Tyrosinemia type I that may also prove to be safer for use in humans. A study published in Nature Biotechnology shows that administering the treatment by combining two delivery mechanisms already in clinical trials for other diseases led to correction of the mutated gene that causes the rare liver disorder in 6 percent of liver cells -- enough to effectively cure the disease in mice. "This is the first study to provide proof that the CRISPR/Cas9 gene editing system can be administered in a therapeutically applicable formulation to repair genes in live, adult animals," said Wen Xue, PhD, assistant professor of molecular medicine and a member of the RNA Therapeutics Institute at UMass Medical School. "Until now it's not been possible to deliver CRISPR/Cas9 in a way that was suitable for clinical trials. By using an RNA guide and DNA repair template delivered via viral vector followed by a Cas9 in a lipid nanoparticle, we've take a huge step forward to overcoming this hurdle." "This finding really excites us because it makes us think that this is a gene repair system that could be used to treat a range of diseases -- not just Tyrosinemia but others as well," said senior author Daniel G. Anderson, PhD, associate professor of chemical engineering at the Massachusetts Institute of Technology and a member of the Koch Institute for Integrative Cancer Research and the Institute for Medical Engineering and Science. CRISPR/Cas9 has become a powerful gene editing tool that is revolutionizing biomedical research by making it easier to inactivate or activate genes in a cell line for study. An adaptive immune system used by bacteria to defend itself against bacteriophage and other types of foreign genetic material, the system consists of two components: a molecular scalpel (Cas9) that cuts DNA and an RNA guide complex that unlocks the scalpel when a matching genetic sequence, defining the exact spot to cut, is found. Scientists can reprogram the CRISPR/Cas9 system with artificial guide RNAs to cleave specific sequences within mammalian genomes. The cell's natural DNA repair processes glue the genome back together, often excising a small portion. If a corrected copy of the disease mutation is also delivered when the cut is made, the cell can stitch the genome back tighter with the corrected gene, leading to permanent repair of the genome and correction of the disease gene. In order to use this technology effectively all three of these elements, including the DNA repair template, must be efficiently and safely delivered to the nuclei of target cells. "Our previous research (published in 2014) showed that it was possible to correct the genetic mutation that causes Tyrosinemia in mice using CRISPR/Cas9 delivered through high-pressured injection," said Dr. Xue. "This approach isn't suitable for clinical applications, though, because it can cause damage to the liver and we'd have to deliver so much of it that we'd be doubling the blood volume." Type 1 Tyrosinemia, also known as hepatorenal tyrosinemia, is caused by the inability to metabolize the amino acid tyrosine. It is caused by a mutation in the FAH gene which codes for the enzyme fumarylacetoacetate hydrolase. This leads to a toxic build-up of metabolites in the blood and urine, causing severe damage to the liver and kidneys. Diagnosed in infants, treatment for the disease includes restriction of tyrosine in the diet, the drug nitisinone and in some cases liver transplant. The challenge for Xue and colleagues was to develop a CRISPR/Cas9 delivery system that was more efficient than the 1 in 250 cells that were repaired via high-pressured injection in the previous study while also being potentially safer for human application. To achieve this, they turned to two genetic delivery systems already in clinical trials--an adeno associated virus (AAV) vector and a lipid nanoparticle. They loaded a CRISPR guide RNA and the genetic repair template into a reprogrammed AAV vector and injected it into mice. Because these genetic materials are being delivered with a viral vector, they can be expressed over a prolonged period of time, alleviating the need to deliver them simultaneously with Cas9. Without the Cas9 messenger RNA to cut the genome, the CRISPR guide and repair template remain inactive in the cells. A week later, after the liver cells have had time to begin producing the RNA guide strand and the DNA template, a lipid nanoparticle is used to deliver the Cas9 messenger RNA. This is a better delivery vehicle for Cas9 RNA because it is typically too big to fit inside an AAV. Additionally, when Cas9 is delivered with a viral vector into the cell, it will continue expressing long after the damaged DNA has been repaired. This increases the likelihood of an off-target edit that could potentially damage the genome. The Cas9 messenger RNA begins to degrade relatively quickly (within days) after delivery and allows for short-term expression of the Cas9. This greatly reduces the risk for potential off target cutting by CRISPR/Cas9 system. When these elements were delivered to adult mice with Tyrosinemia type I, the animals experienced a reduction of weight loss, alleviation of liver damage and generation of liver cells with the corrected FAH gene. Deep sequencing of the treated liver cells revealed that 6 percent of them harbored the corrected FAH gene--about 1 in 16 or a 15-fold improvement over the previous study. It also reduced off-target cutting of the genome by seven-fold. "We combined the clinical suitable non-viral and viral delivery systems to allow efficiently gene repair in vivo, and to minimize the side effects," said lead author Hao Yin, PhD, research scientist at MIT. Because AAV vectors are already in clinical trials for other human disorders and Dr. Anderson has similar lipid nanoparticle in clinical development, the researchers are optimistic that this CRISPR delivery method could be used in humans, although more studies are needed. "The hope is that because we've used two delivery methods already in clinical development for patients, it will expedite a path to clinical trials for a CRISPR treatment of Tyrosinemia type I," said Xue. "What's more, the platform we've devised is modular, so it can potentially be tailored to treat other diseases, especially in the liver." About University of Massachusetts Medical School The University of Massachusetts Medical School, one of the fastest growing academic health centers in the country, has built a reputation as a world-class research institution, consistently producing noteworthy advances in clinical and basic research. The Medical School attracts more than $277 million in research funding annually, 80 percent of which comes from federal funding sources. The mission of the Medical School is to advance the health and well-being of the people of the commonwealth and the world through pioneering education, research, public service and health care delivery with its clinical partner, UMass Memorial Health Care. For more information, please click If you have a comment, please us. Issuers of news releases, not 7th Wave, Inc. or Nanotechnology Now, are solely responsible for the accuracy of the content.

Hauspurg A.K.,Yale University | Allred E.N.,Neuroepidemiology Unit | Allred E.N.,Harvard University | Vanderveen D.K.,Harvard University | And 9 more authors.
Neonatology | Year: 2011

Objective: This study tested the hypothesis that preterm infants who had a blood gas derangement on at least 2 of the first 3 postnatal days are at increased risk for more severe retinopathy of prematurity (ROP). Method: 1,042 infants born before 28 weeks' gestational age (GA) were included. An infant was considered to be exposed if his/her blood gas measure was in the highest or lowest quartile for GA on at least 2 of the first 3 postnatal days. Results: Multivariable models adjusting for confounders indicate that exposure to a PCO2 in the highest quartile predicts ROP (stage 3, 4 or 5: OR = 1.6, 95% CI = 1.1-2.3); zone 1: 2.0, 1.1-3.6; prethreshold/threshold: 1.9, 1.2-3.0; plus disease: 1.8, 1.1-2.9). Estimates are similar for a low pH for zone 1 (2.1, 1.2-3.8), prethreshold/threshold (1.8, 1.1-2.8), but did not quite achieve statistical significance for ROP stage 3, 4, or 5 (1.4, 0.9-2.0) and plus disease (1.5, 0.9-2.4). A PaO2 in the highest quartile for GA on at least 2 of the first 3 postnatal days was associated with a doubling of the risk of ROP in zone 1 (2.5, 1.4-4.4) and of prethreshold/threshold disease (2.1, 1.4-3.3), a 70% risk increase for plus disease (1.7, 1.04-2.8), while a 40% risk increase for ROP stage 3 or higher did not achieve statistical significance (1.4, 0.96-2.0). Conclusion: Infants exposed to high PCO2, low pH and high PaO2 appear to be at increased risk of more severe ROP. Copyright © 2010 S. Karger AG, Basel.

Hingorani A.,Lutheran Medical Center | Lamuraglia G.M.,Harvard University | Henke P.,University of Michigan | Meissner M.H.,University of Washington | And 8 more authors.
Journal of Vascular Surgery | Year: 2016

Background Diabetes mellitus continues to grow in global prevalence and to consume an increasing amount of health care resources. One of the key areas of morbidity associated with diabetes is the diabetic foot. To improve the care of patients with diabetic foot and to provide an evidence-based multidisciplinary management approach, the Society for Vascular Surgery in collaboration with the American Podiatric Medical Association and the Society for Vascular Medicine developed this clinical practice guideline. Methods The committee made specific practice recommendations using the Grades of Recommendation Assessment, Development, and Evaluation system. This was based on five systematic reviews of the literature. Specific areas of focus included (1) prevention of diabetic foot ulceration, (2) off-loading, (3) diagnosis of osteomyelitis, (4) wound care, and (5) peripheral arterial disease. Results Although we identified only limited high-quality evidence for many of the critical questions, we used the best available evidence and considered the patients' values and preferences and the clinical context to develop these guidelines. We include preventive recommendations such as those for adequate glycemic control, periodic foot inspection, and patient and family education. We recommend using custom therapeutic footwear in high-risk diabetic patients, including those with significant neuropathy, foot deformities, or previous amputation. In patients with plantar diabetic foot ulcer (DFU), we recommend off-loading with a total contact cast or irremovable fixed ankle walking boot. In patients with a new DFU, we recommend probe to bone test and plain films to be followed by magnetic resonance imaging if a soft tissue abscess or osteomyelitis is suspected. We provide recommendations on comprehensive wound care and various débridement methods. For DFUs that fail to improve (>50% wound area reduction) after a minimum of 4 weeks of standard wound therapy, we recommend adjunctive wound therapy options. In patients with DFU who have peripheral arterial disease, we recommend revascularization by either surgical bypass or endovascular therapy. Conclusions Whereas these guidelines have addressed five key areas in the care of DFUs, they do not cover all the aspects of this complex condition. Going forward as future evidence accumulates, we plan to update our recommendations accordingly. © 2016 Society for Vascular Surgery.

Juhascik M.,UMass Memorial | Gleba J.,UMass Memorial | Jenkins A.,UMass Memorial
Journal of Analytical Toxicology | Year: 2012

In forensic toxicology general alkaline drug screens typically utilize liquid liquid [LLE] or solid phase extraction [SPE] sample preparation techniques. It is expected that different drugs will be detected when a laboratory changes techniques. In this study, when the authors switched from LLE to SPE they were able to detect benzoylecgonine [BE]. Benzoylecgonine isopropyl ester [BEIE] was also detected. Further investigation demonstrated that the BEIE was formed during sample elution with methylene chloride/isopropanol/ammonium hydroxide. BEIE was not detected if methanol/ammonium hydroxide was used as the elution solvent. Analysts should be aware that BEIE is formed in the presence of BE if elution solvents comprise isopropanol and a strong base. Therefore, use of BEIE as an internal standard in such assays will result in inaccurate quantitation of BE. © The Author [2012]. Published by Oxford University Press. All rights reserved.

Tocci E.M.,University of Massachusetts Medical School | Robinson A.,UMass Memorial | Belazarian L.,UMass Memorial | Belazarian L.,University of Massachusetts Medical School | And 4 more authors.
Pediatric Dermatology | Year: 2015

Propylene glycol is a well-documented causative agent of allergic contact dermatitis (ACD). It is also reported to cause systemic dermatitis after ingestion of foods or medicines containing it and after intravenous injection of a medicine with propylene glycol in its base. We describe two adolescents with sensitivity to propylene glycol confirmed by patch testing whose dermatitis improved dramatically after cessation of oral antihistamines containing propylene glycol. We report these cases to alert providers to the potential for worsening of ACD due to systemic exposure to propylene glycol in patients with a cutaneous sensitivity to the allergen. © 2015 Wiley Periodicals, Inc.

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