Although cord blood transplantation has significantly extended the lifespan of mucopolysaccharidosis type 1 (MPS1) patients, over 95% manifest cornea clouding with about 50% progressing to blindness. As corneal transplants are met with high rejection rates in MPS1 children, there remains no treatment to prevent blindness or restore vision in MPS1 children. Since MPS1 is caused by mutations in idua, which encodes alpha-L-iduronidase, a gene addition strategy to prevent, and potentially reverse, MPS1-associated corneal blindness was investigated. Initially, a codon optimized idua cDNA expression cassette (opt-IDUA) was validated for IDUA production and function following adeno-associated virus (AAV) vector transduction of MPS1 patient fibroblasts. Then, an AAV serotype evaluation in human cornea explants identified an AAV8 and 9 chimeric capsid (8G9) as most efficient for transduction. AAV8G9-opt-IDUA administered to human corneas via intrastromal injection demonstrated widespread transduction, which included cells that naturally produce IDUA, and resulted in a >10-fold supraphysiological increase in IDUA activity. No significant apoptosis related to AAV vectors or IDUA was observed under any conditions in both human corneas and MPS1 patient fibroblasts. The collective preclinical data demonstrate safe and efficient IDUA delivery to human corneas, which may prevent and potentially reverse MPS1-associated cornea blindness.
Many adeno-associated virus (AAV) serotypes efficiently transduce the retina when delivered to the subretinal space but show limited success when delivered to the vitreous due to the inner limiting membrane (ILM). Subretinal delivery of AAV serotype 2 (AAV2) and its heparan sulfate (HS)-binding-deficient capsid led to similar expression, indicating transduction of the outer retina occurred by HS-independent mechanisms. However, intravitreal delivery of HS-ablated recombinant AAV2 (rAAV2) led to a 300-fold decrease in transduction compared to AAV2. Fluorescence in situ hybridization of AAV transgenes was used to identify differences in retinal trafficking and revealed that HS binding was responsible for AAV2 accumulation at the ILM. This mechanism was tested on human ex vivo retinas and showed similar accumulation with HS-binding AAV2 capsid only. To evaluate if HS binding could be applied to other AAV serotypes to enhance their transduction, AAV1 and AAV8 were modified to bind HS with a single-amino-acid mutation and tested in mice. Both HS-binding mutants of AAV1 and AAV8 had higher intravitreal transduction than their non-HS-binding parent capsid due to increased retinal accumulation. To understand the influence that HS binding has on tropism, chimeric AAV2 capsids with dual-glycan usage were tested intravitreally in mice. Compared to HS binding alone, these chimeric capsids displayed enhanced transduction that was correlated with a change in tropism. Taken together, these data indicate that HS binding serves to sequester AAV capsids from the vitreous to the ILM but does not influence retinal tropism. The enhanced retinal transduction of HS-binding capsids provides a rational design strategy for engineering capsids for intravitreal delivery. A deno-associated virus (AAV) is a small (25-nm), nonpathogenic virus that has been extensively studied as a vector for gene transfer applications (1-3). The virus consists of two parts: the viral genome and the protein capsid. The viral genome can be largely replaced with a desired transgene to create recombinant AAV (rAAV) vectors used for gene delivery. The protein capsid is responsible for cell attachment and entry via a variety of glycans and cell surface receptors. There are 11 naturally occurring serotypes of AAV, denoted AAV1 to AAV11. Glycans and receptors have been elucidated for several AAV serotypes. Heparan sulfate proteoglycan (HSPG) has been shown to be used for both rAAV2 and rAAV3 cell entry (4). rAAV6 displays dual-glycan interaction with HSPG and sialic acid; however, HSPG binding alone is insufficient for cellular entry (5). Various linkages of sialic acid are important for the transduction of the rAAV1, rAAV4, and rAAV5 serotypes (6,7). N-linked galactose is used for the transduction of the rAAV9 serotype (8). Glycans expressed on the cell surface dictate the tissue and cellular tropism observed with the various AAV capsids. In addition to attachment to these glycans, AAV serotypes interact with cell receptors, including human growth factor rec...
Background Types and correlates of pain medication agreement (PMA) violations in the primary care setting have not been analyzed. Methods A retrospective analysis was completed to examine patient characteristics and correlates of PMA violations, a proxy for substance misuse, over a 15-year period in an outpatient General Medicine Pain Service within the Division of General Medicine and Clinical Epidemiology, Department of Medicine, School of Medicine, University of North Carolina at Chapel Hill. Patients who signed the PMA were managed for chronic pain from 2002 through 2017 (N = 1,210). The incidence of PMA violations was measured over a 15-year span. Substance misuse was defined a priori in the study as urine toxicology screen positive for illicit or nonprescribed controlled substances, patient engagement in prescription alteration, doctor-shopping, or diversion. Results Most patients received a prescription for a controlled substance (77.4%). During enrollment, 488 (40.3%) patients had one or more violations of their PMA. One-third (33.4%) of pain service patients had a violation within 365 days of signing the agreement. Active tobacco smokers had double the incidence of agreement violation within the first 30 days of enrollment. Almost one-half (49.8%) of violations were due to inconsistent use of controlled substances. Patients with any prior DWI/DUI or drug-related offense had a significantly increased rate of substance misuse (P < 0.0001). Conclusions PMA violations were common among a population of patients managed for chronic nonmalignant pain. Universal opioid prescribing precautions, including PMAs, require further investigation to assess their roles in mitigating the potential patient and societal harms associated with opioid prescribing.
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