Increasing the Specificity of AAV-Based Gene Editing through Self-Targeting and Short-Promoter Strategies.

Our group previously used adeno-associated viral vectors (AAVs) to express an engineered meganuclease specific for a sequence in the PCSK9 gene (M2PCSK9), a clinical target for treating coronary heart disease. Upon testing this nuclease in non-human primates, we observed specific editing characterized by several insertions and deletions (indels) in the target sequence as well as indels in similar genomic sequences. We hypothesized that high nuclease expression increases off-target editing. Here, we reduced nuclease expression using two strategies. The first was a self-targeting strategy that involved inserting the M2PCSK9 target sequence into the AAV genome that expresses the nuclease and/or fusing the nuclease to a specific peptide to promote its degradation. The second strategy used a shortened version of the parental promoter to reduce nuclease expression. Mice administered with these second-generation AAV vectors showed reduced PCSK9 expression due to the nuclease on-target activity and reduced off-target activity. All vectors induced a stable reduction of PCSK9 in primates treated with self-targeting and short-promoter AAVs. Compared to the meganuclease-expressing parental AAV vector, we observed a significant reduction in off-target activity. In conclusion, we increased the in vivo nuclease specificity using a clinically relevant strategy that can be applied to other genome-editing nucleases.

Muscle-directed AAV gene therapy rescues the maple syrup urine disease phenotype in a mouse model.

Maple syrup urine disease (MSUD) is a rare, inherited metabolic disorder characterized by a dysfunctional mitochondrial enzyme complex, branched-chain alpha-keto acid dehydrogenase (BCKDH), which catabolizes branched-chain amino acids (BCAAs). Without functional BCKDH, BCAAs and their neurotoxic alpha-keto intermediates can accumulate in the blood and tissues. MSUD is currently incurable and treatment is limited to dietary restriction or liver transplantation, meaning there is a great need to develop new treatments for MSUD. We evaluated potential gene therapy applications for MSUD in the intermediate MSUD (iMSUD) mouse model, which harbors a mutation in the dihydrolipoamide branched-chain transacylase E2 (DBT) subunit of BCKDH. Systemic delivery of an adeno-associated virus (AAV) vector expressing DBT under control of the liver-specific TBG promoter to the liver did not sufficiently ameliorate all aspects of the disease phenotype. These findings necessitated an alternative therapeutic strategy. Muscle makes a larger contribution to BCAA metabolism than liver in humans, but a muscle-specific approach involving a muscle-specific promoter for DBT expression delivered via intramuscular (IM) administration only partially rescued the MSUD phenotype in mice. Combining the muscle-tropic AAV9 capsid with the ubiquitous CB7 promoter via IM or IV injection, however, substantially increased survival across all assessed doses. Additionally, near-normal serum BCAA levels were achieved and maintained in the mid- and high-dose cohorts throughout the study; this approach also protected these mice from a lethal high-protein diet challenge. Therefore, administration of a gene therapy vector that expresses in both muscle and liver may represent a viable approach to treating patients with MSUD.

Lipid nanoparticle-encapsulated mRNA therapy corrects serum total bilirubin level in Crigler-Najjar syndrome mouse model.

Crigler-Najjar syndrome is a rare disorder of bilirubin metabolism caused by uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) mutations characterized by hyperbilirubinemia and jaundice. No cure currently exists; treatment options are limited to phototherapy, whose effectiveness diminishes over time, and liver transplantation. Here, we evaluated the therapeutic potential of systemically administered, lipid nanoparticle-encapsulated human UGT1A1 (hUGT1A1) mRNA therapy in a Crigler-Najjar mouse model. Ugt1 knockout mice were rescued from lethal post-natal hyperbilirubinemia by phototherapy. These adult Ugt1 knockout mice were then administered a single lipid nanoparticle-encapsulated hUGT1A1 mRNA dose. Within 24 h, serum total bilirubin levels decreased from 15 mg/dL (256 µmol/L) to <0.5 mg/dL (9 µmol/L), i.e., slightly above wild-type levels. This reduction was sustained for 2 weeks before bilirubin levels rose and returned to pre-treatment levels by day 42 post-administration. Sustained reductions in total bilirubin levels were achieved by repeated administration of the mRNA product in a frequency-dependent manner. We were also able to rescue the neonatal lethality phenotype seen in Ugt1 knockout mice with a single lipid nanoparticle dose, which suggests that this may be a treatment modality appropriate for metabolic crisis situations. Therefore, lipid nanoparticle-encapsulated hUGT1A1 mRNA may represent a potential treatment for Crigler-Najjar syndrome.

Determining the Minimally Effective Dose of a Clinical Candidate Adeno-Associated Virus Vector in a Mouse Model of Hemophilia A.

Hemophilia A, a bleeding disorder, affects 1:5,000 males and is caused by a deficiency of human blood coagulation factor VIII (hFVIII). Studies in mice and macaques identified AAVhu37.E03.TTR.hFVIIIco-SQ.PA75 as a clinical candidate gene therapy vector to treat hemophilia A. In this study, we sought to determine the minimally effective dose (MED) of this vector in a hemophilia A mouse model. Mice received one of four vector doses (3 × 1011-1 × 1013 genome copies [GCs]/kg) via intravenous tail vein injection; one cohort received vehicle as a control. Animals were monitored daily after vector/vehicle administration. Blood samples were collected to evaluate hFVIII activity levels and anti-hFVIII antibodies. Animals were sacrificed and necropsied on days 28 and 56; tissues were harvested for histopathological examination and blood was collected for serum chemistry panel analysis. We found no significant differences in liver transaminase levels in mice administered any vector dose compared to those administered vehicle (except for one group administered 3 × 1011 GC/kg). Total bilirubin levels were significantly elevated compared to the vehicle group following two vector doses at day 56 (1 × 1012 and 1 × 1013 GC/kg). We observed no vector-related gross or histological findings. Most microscopic findings were in the vehicle group and considered secondary to blood loss, an expected phenotype of this mouse model. Since we observed no dose-limiting safety markers, we determined that the maximally tolerated dose was greater than or equal to the highest dose tested (1 × 1013 GC/kg). Since we detected hFVIII activity in all cohorts administered vector, we conclude that the MED is 3 × 1011 GC/kg-the lowest dose evaluated in this study.

Treating Transthyretin Amyloidosis via Adeno-Associated Virus Vector Delivery of Meganucleases.

Transthyretin amyloidosis (ATTR) is a progressive and fatal disease caused by transthyretin (TTR) amyloid fibril accumulation in tissues, which disrupts organ function. As the TTR protein is primarily synthesized by the liver, liver transplantation can cure familial ATTR but is not an option for the predominant age-related wild-type ATTR. Approved treatment approaches include TTR stabilizers and an RNA-interference therapeutic, but these require regular re-administration. Gene editing could represent an effective one-time treatment. We evaluated adeno-associated virus (AAV) vector-delivered, gene-editing meganucleases to reduce TTR levels. We used engineered meganucleases targeting two different sites within the TTR gene. AAV vectors expressing TTR meganuclease transgenes were first tested in immunodeficient mice expressing the human TTR sequence delivered using an AAV vector and then against the endogenous TTR gene in rhesus macaques. Following a dose of 3 × 1013 genome copies per kilogram, we detected on-target editing efficiency of up to 45% insertions and deletions (indels) in the TTR genomic DNA locus and >80% indels in TTR RNA, with a concomitant decrease in serum TTR levels of >95% in macaques. The significant reduction in serum TTR levels following TTR gene editing indicates that this approach could be an effective treatment for ATTR.

A Gene Therapy Approach to Improve Copper Metabolism and Prevent Liver Damage in a Mouse Model of Wilson Disease.

Wilson disease (WD), an autosomal recessive disease caused by mutations in a copper-transporting P-type ATPase (Atp7b), causes severe liver damage. This disease is currently treated with the lifelong use of copper chelation therapy, which has side effects and does not fix copper metabolism. Here, we thoroughly characterized a mouse model of WD, the toxic milk mouse, and used the model to test a gene therapy approach for treating WD. WD mice accumulated copper in the liver from birth; severe copper accumulation and concurrent liver disease were evident by 2 months of age. Intravenously administering an adeno-associated viral (AAV) 8 vector expressing a codon-optimized version of the human ATP7B transgene into 2-month-old WD mice significantly decreased liver copper levels compared with age-matched, uninjected, WD mice. We also observed a significant dose-dependent decrease in liver disease. Male mice injected with 1011 genome copies of AAV8 vector showed only mild histopathological findings with a complete lack of liver fibrosis. Therefore, we conclude that administering gene therapy at the early stages of disease onset is a promising approach for reducing liver damage and correcting copper metabolism in WD.

The implications of gender stereotypes for the dentist-patient relationship.

This study assessed whether traditionally based gender stereotypes are applied to dentists. Awareness of gender-driven preconceptions can help dentists anticipate patient expectations that play a role in the clinician-patient relationship. A sample of 106 college students and fifty-four non-college students completed a one-page survey that assessed whether seven traits were viewed as more characteristic of male dentists, female dentists, or neither gender. While there was no trait that over 50 percent of respondents considered more typical of either gender, female dentists were viewed as significantly more likely to make patients feel relaxed and to take time to discuss ailments with them, perceptions found most frequently among those respondents who expressed a preference for a female dentist. Male dentists were perceived as significantly more likely to expect a patient to endure pain without complaints, more devoted to career than family, and more likely to seem to be in charge and to be attracted to the power of their profession. The findings indicate that dental students should be encouraged to consider how patients' preconceived gender-based expectations and assumptions may influence rapport and communication between patients and dentists.

Early, focal changes in cartilage cellularity and structure following surgically induced meniscal destabilization in the mouse.

Post-traumatic osteoarthritis (PTOA) is an accelerated form of osteoarthritic cartilage degeneration affecting approximately 20-50% of patients experiencing joint injury. Currently PTOA is incurable; to better understand the etiology of PTOA and to develop rational anti-osteoarthritic therapies, it is critical to understand the spatiotemporal initiation and the progression of PTOA. In this study, we employed semi-quantitative histological scoring and quantitative damage analysis to examine disease progression in the murine destabilization of the medial meniscus (DMM) model of PTOA from early (3 days) through late- (112 days) disease timepoints. We observed significant, progressive articular cartilage (AC) cellular, and structural changes in the medial compartments of injured joints as early as 3 days. Spatially within the joint, cartilage damage (erosions) were observed anteriorly at 84 days. Furthermore, a drastic loss in chondrocyte number (by 3 days), surface damage (at 7 days), and cartilage erosion (at 84 days) was found to co-localize to the specific region of the medial tibial plateau AC that experienced a change in meniscal coverage due to meniscal extrusion following DMM. Taken together, these results suggest that DMM-mediated extrusion of the medial meniscus leads to rapid, spatially dependent changes in AC cellularity and structure, and precipitates the focal degeneration of cartilage associated with PTOA. Importantly, this study suggests that joint instability injuries may trigger immediate (<3 days) processes within a small population of chondrocytes that directs the initiation and progression of PTOA, and that development of chondroprotective strategies for preventing and/or delaying PTOA-related cartilage degeneration are best targeted toward these immediately early processes following joint injury. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:537-547, 2017.

Seeing through Musculoskeletal Tissues: Improving In Situ Imaging of Bone and the Lacunar Canalicular System through Optical Clearing.

In situ, cells of the musculoskeletal system reside within complex and often interconnected 3-D environments. Key to better understanding how 3-D tissue and cellular environments regulate musculoskeletal physiology, homeostasis, and health is the use of robust methodologies for directly visualizing cell-cell and cell-matrix architecture in situ. However, the use of standard optical imaging techniques is often of limited utility in deep imaging of intact musculoskeletal tissues due to the highly scattering nature of biological tissues. Drawing inspiration from recent developments in the deep-tissue imaging field, we describe the application of immersion based optical clearing techniques, which utilize the principle of refractive index (RI) matching between the clearing/mounting media and tissue under observation, to improve the deep, in situ imaging of musculoskeletal tissues. To date, few optical clearing techniques have been applied specifically to musculoskeletal tissues, and a systematic comparison of the clearing ability of optical clearing agents in musculoskeletal tissues has yet to be fully demonstrated. In this study we tested the ability of eight different aqueous and non-aqueous clearing agents, with RIs ranging from 1.45 to 1.56, to optically clear murine knee joints and cortical bone. We demonstrated and quantified the ability of these optical clearing agents to clear musculoskeletal tissues and improve both macro- and micro-scale imaging of musculoskeletal tissue across several imaging modalities (stereomicroscopy, spectroscopy, and one-, and two-photon confocal microscopy) and investigational techniques (dynamic bone labeling and en bloc tissue staining). Based upon these findings we believe that optical clearing, in combination with advanced imaging techniques, has the potential to complement classical musculoskeletal analysis techniques; opening the door for improved in situ investigation and quantification of musculoskeletal tissues.