Survey of Pharmaceutical Industry's Best Practices around In Vitro Transporter Assessment and Implications for Drug Development: Considerations from the International Consortium for Innovation and Quality for Pharmaceutical Development Transporter Working Group.

The International Consortium for Innovation and Quality in Pharmaceutical Development Transporter Working Group had a rare opportunity to analyze a crosspharma collation of in vitro data and assay methods for the evaluation of drug transporter substrate and inhibitor potential. Experiments were generally performed in accordance with regulatory guidelines. Discrepancies, such as not considering the impact of preincubation for inhibition and free or measured in vitro drug concentrations, may be due to the retrospective nature of the dataset and analysis. Lipophilicity was a frequent indicator of crosstransport inhibition (P-gp, BCRP, OATP1B, and OCT1), with high molecular weight (MW ≥500 Da) also common for OATP1B and BCRP inhibitors. A high level of overlap in in vitro inhibition across transporters was identified for BCRP, OATP1B1, and MATE1, suggesting that prediction of DDIs for these transporters will be common. In contrast, inhibition of OAT1 did not coincide with inhibition of any other transporter. Neutrals, bases, and compounds with intermediate-high lipophilicity tended to be P-gp and/or BCRP substrates, whereas compounds with MW <500 Da tended to be OAT3 substrates. Interestingly, the majority of in vitro inhibitors were not reported to be followed up with a clinical study by the submitting company, whereas those compounds identified as substrates generally were. Approaches to metabolite testing were generally found to be similar to parent testing, with metabolites generally being equally or less potent than parent compounds. However, examples where metabolites inhibited transporters in vitro were identified, supporting the regulatory requirement for in vitro testing of metabolites to enable integrated clinical DDI risk assessment. SIGNIFICANCE STATEMENT: A diverse dataset showed that transporter inhibition often correlated with lipophilicity and molecular weight (>500 Da). Overlapping transporter inhibition was identified, particularly that inhibition of BCRP, OATP1B1, and MATE1 was frequent if the compound inhibited other transporters. In contrast, inhibition of OAT1 did not correlate with the other drug transporters tested.

Dupilumab pharmacokinetics and effect on type 2 biomarkers in children with moderate-to-severe asthma.

BACKGROUND: Type 2 inflammation is common in children with asthma. Dupilumab, a human antibody, blocks the signaling of interleukin -4 and -13, key and central drivers of type 2 inflammation. In the LIBERTY ASTHMA VOYAGE (NCT02948959) study, dupilumab reduced severe asthma exacerbations and improved lung function in children aged 6 to 11 years with uncontrolled, moderate-to-severe asthma. OBJECTIVE: To assess the pharmacokinetics of dupilumab and type 2 biomarker changes in children with type 2 asthma in VOYAGE. METHODS: Patients were randomized to dupilumab 100 mg (≤30 kg) or 200 mg (>30 kg) or placebo every 2 weeks for 52 weeks. Dupilumab concentrations and changes in type 2 biomarkers were assessed at each visit. RESULTS: Dupilumab concentrations in serum reached a steady state by week 12, with mean concentrations of 51.2 mg/L and 79.4 mg/L in children receiving dupilumab 100 mg every 2 weeks and 200 mg every 2 weeks, respectively (therapeutic range in adults and adolescents: 29-80 mg/L). Reductions in type 2 biomarkers were comparable between regimens, and greater in patients treated with dupilumab vs placebo. In children treated with dupilumab 100 mg and 200 mg every 2 weeks, the median percent changes (Q1-Q3) from baseline at week 52 were, respectively, -78.6% (-86.3 to -69.80) and -78.6% (-84.9 to -70.1) for serum total immunoglobulin E, -53.6% (-66.4 to -34.6) and -43.7% (-58.6 to -28.5) for thymus and activation-regulated chemokine; -25.7% (-60.0 to 27.6) and -33.3% (-60.6 to 16.6) for blood eosinophils, and -47.7% (-73.8 to 18.9) and -55.6% (-73.6 to -20.0) for fractional exhaled nitric oxide. CONCLUSION: Weight-tiered dose regimens achieved mean concentrations within the dupilumab therapeutic range. The median decreases in type 2 biomarker levels were similar between dose regimens. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02948959.

Pharmacokinetics, pharmacodynamics, and safety of single-dose subcutaneous sarilumab with or without methotrexate in Japanese patients with rheumatoid arthritis: Two single-dose studies.

OBJECTIVES: To assess the safety and pharmacokinetics (PK) of single-dose subcutaneous (SC) sarilumab or tocilizumab SC ± methotrexate (MTX) and to assess the pharmacodynamics (PD) of sarilumab SC or tocilizumab SC monotherapy in Japanese rheumatoid arthritis (RA) patients. METHODS: TDU13402 was a randomized, double-blind, placebo-controlled, single-ascending dose Phase 1 study (NCT01850680). Twenty-four patients (6 per treatment group) received sarilumab 50, 100, or 200 mg plus MTX or placebo (2 per cohort) on Day (D) 1; PK and safety were assessed through D57. PDY14191 was a randomized, open-label, single-dose study (NCT02404558). Thirty patients (15 per arm) received sarilumab 150 mg or tocilizumab 162 mg on D1; PK, PD, and safety were assessed through D43. RESULTS: TDU13402: mean serum sarilumab exposure increased in a greater than dose proportional manner from 50 to 200 mg dose with no clinically meaningful increase in treatment-emergent adverse events (TEAEs). PDY14191: PK profiles of single-dose sarilumab 150 mg or tocilizumab 162 mg were similar; some numerical differences in PD profiles and TEAEs were observed. Neutrophil count decrease/neutropenia was the most frequently reported TEAE with sarilumab treatment in both studies. CONCLUSIONS: PK, PD, and safety profiles of single-dose sarilumab SC with/without MTX were consistent with results anticipated in Japanese patients with RA.

Oxytocin receptor behavioral effects and cell types in the bed nucleus of the stria terminalis.

Oxytocin is a neuropeptide that can produce anxiolytic effects and promote social approach. However, emerging evidence shows that under some conditions, oxytocin can instead induce anxiety-related behaviors. These diverse effects of oxytocin appear to be mediated by circuit-specific actions. Recent data showed that inhibition of oxytocin receptors (OTRs) in the bed nucleus of the stria terminalis (BNST) was sufficient to increase social approach and decrease social vigilance in female California mice (Peromyscus californicus) exposed to social defeat stress. As a member of the G-protein coupled receptor family, OTRs can induce distinct downstream pathways by coupling to different G-protein isoforms. We show that infusion of carbetocin, a biased OTR-Gq agonist, in the BNST reduced social approach in both female and male California mice. In both females and males, carbetocin also increased social vigilance. To gain insight into cell types that could be mediating this effect, we analyzed previously published single-cell RNAseq data from the BNST and nucleus accumbens (NAc). In the NAc, we and others showed that OTR activation promotes social approach behaviors. In the BNST, Oxtr was expressed in over 40 cell types, that span both posterior and anterior subregions of the BNST. The majority of Oxtr-expressing neurons were GABAergic. In the anterior regions of BNST targeted in our carbetocin experiments, Cyp26b1-expressing neurons had high average Oxtr expression. In the NAc, most Oxtr+ cells were D1 dopamine receptor-expressing neurons and interneurons. These differences in Oxtr cell type distribution may help explain how activation of OTR in BNST versus NAc can have different effects on social approach and social vigilance.

REPRODUCTIVE OPHTHALMOLOGY: The Intersection of Inherited Eye Diseases and Reproductive Technologies.

PURPOSE: To propose a working framework for patients with inherited eye diseases presenting to ophthalmologists who are interested in assisted reproductive technology and preimplantation genetic testing. METHODS: Retrospective chart review and case series of three families with inherited eye diseases who successfully underwent preimplantation genetic testing, in vitro fertilization, and birth of unaffected children. RESULTS: Preimplantation genetic testing was performed for three families with different inherited eye diseases, which included autosomal dominant retinitis pigmentosa, autosomal recessive achromatopsia, and X-linked Goltz syndrome. Preimplantation genetic testing led to the identification of unaffected embryos, which were then selected for in vitro fertilization and resulted in the birth of unaffected children. CONCLUSION: A close collaboration between patients, families, ophthalmologists, reproductive genetic counselors, and reproductive endocrinology and infertility specialists is the ideal model for taking care of patients interested in preimplantation genetic testing for preventing the transmission of inherited eye diseases.

Immunogenicity of sarilumab and impact on safety and efficacy in Japanese patients with rheumatoid arthritis: analysis of two Phase 3 randomised clinical trials.

OBJECTIVES: To describe the immunogenicity profile of sarilumab in Japanese patients with rheumatoid arthritis (RA). METHODS: Patients enrolled in the KAKEHASI and HARUKA studies were included in our analysis. In these studies, patients received sarilumab 150 mg or 200 mg every 2 weeks for 52 or 28 weeks in combination with methotrexate (MTX) (KAKEHASI), or for 52 weeks as monotherapy or in combination with non-MTX conventional synthetic disease-modifying anti-rheumatic drugs (HARUKA). Anti-drug antibodies (ADAs) and neutralising antibodies (NAbs) were assessed in the pooled population. RESULTS: Positive ADA assay responses occurred in 10/149 (7.1%) patients treated with sarilumab 150 mg and 13/185 (7.0%) patients treated with sarilumab 200 mg, with persistent responses in 2 (1.4%) and 4 (2.2%) patients, respectively. Peak ADA titre was 30. No patients treated with the 150 mg dose and one patient (0.5%) treated with the 200 mg dose exhibited NAbs. There was no evidence of an association between ADA formation and hypersensitivity reactions or reduced efficacy. CONCLUSIONS: ADAs, which occurred at a low frequency and titre, did not affect the safety or efficacy of sarilumab 150 or 200 mg administered as monotherapy or combination therapy in Japanese patients with RA in the KAKEHASI or HARUKA studies.

The impact of discomfort with HIV status and hopelessness on depressive symptoms among adolescents living with HIV in Uganda.

Introduction: Discomfort with and reluctance to disclose HIV status can lead to depression, social isolation, and poor medication adherence; we examined relationships among these variables within a sample of adolescents living with HIV in Uganda. Methods: We used baseline data from the Suubi + Adherence study, which recruited a total of 702 adolescents (ages 10-16 years) living with HIV in southwestern Uganda. Structural equation models were conducted separately among in-school adolescents and out-of-school adolescents to assess associations between discomfort level with HIV status and depressive symptoms and if this association was mediated by hopelessness. Results: Out-of-school adolescents had significantly higher depression scores compared with those in-school youth (M = 6.24 vs. M = 5.03, p < 0.001). Although high discomfort level with HIV status was significantly associated with higher depression scores among both in-school adolescents and out-of-school adolescents, this association among out-of-school adolescents (B = 0.49, 95%CI: 0.19, 0.79), was more substantial than for in-school adolescents (B = 0.10, 95%CI: 0.03, 0.17). Discussion: One's discomfort with their HIV status and the extent to which they are hopeful about the future can contribute to the development of depressive symptoms and these factors need to be considered in the development of assessments and interventions for the treatment of depression among adolescents living with HIV.

Understanding barriers to treatment among individuals not engaged in treatment who misuse opioids: A structural equation modeling approach.

Background: Many individuals misusing opioids do not enter into treatment. The question of who enters into treatment for their opioid abuse and under what circumstances is complex and shaped by multiple factors. The objective of the current study is to explore the risk factors for wide-ranging and numerous barriers to treatment among social media users. Method: Opioid-related forums within a popular social media platform were used to recruit non-treatment engaged individuals (≥15 years) who had misused opioids in the past month (n = 144; 66% male; median age 28). Four treatment barrier factors were identified utilizing principle component analysis: (1) stigma, (2) awareness, (3) attitudinal, and (4) denial. A structural equation model (SEM) was then created to explore the risk factors for different types of barriers to OUD treatment. Results: The most common barriers among participants not engaged in treatment for their opioid misuse were the belief that one should be able to help themselves with their condition (66%), treatment was too expensive (63%), and worries about being labeled or judged (57%). Additionally, SEM results demonstrate stigma barriers, awareness, and attitudinal barriers were associated with mental health comorbidities, opioid abuse and dependence severity, and treatment history. Denial barriers, however, were only associated with treatment history, and structural/financial barriers were only associated with opioid abuse and dependence severity. Conclusions: Our research findings are relevant for underscoring the wide-ranging and numerous barriers to treatment faced by individuals misusing opioids that are especially concentrated among those who also struggle with comorbid mental illness.

Leveraging social media to explore the barriers to treatment among individuals with depressive symptoms.

BACKGROUND: Individuals with depression may not seek treatment for their symptoms due to several types of barriers to treatment. In support of the growing research on mental health care access and the role of social media, this study aimed to increase knowledge of these barriers among social media users. METHODS: Participants were recruited from several social media platforms, including Instagram, Facebook, Twitter, Reddit, Tumblr, and online depression forums. Eligible participants had endorsed having posted about feeling sad or depressed on social media, or followed social media groups that post about depression-related topics. Participants completed an online survey about their depression symptoms, interest in treatment, and potential barriers to accessing treatment. RESULTS: Of the participants reaching criteria for depression, those with major depression were more likely to seek out treatment, to report an unmet need for treatment, and have a higher risk of suicide. For participants with major depression, barriers to treatment were more likely to be attitudinal, while participants with mild depression experienced more structural barriers. CONCLUSIONS: This study demonstrates several barriers to treatment that occur for individuals struggling with depression, and that online platforms are effective mediums to recruit individuals with depression symptoms who seek mental health support.

Social and Economic Equity and Family Cohesion as Potential Protective Factors from Depression Among Adolescents Living with HIV in Uganda.

Adolescents living with HIV in Uganda are impacted by poverty and face a number of health and social challenges including access to medication, health complications, and social stigma. These stressors have been linked to depression, which can lead to lower HIV treatment adherence. This study seeks to determine how social and economic equity, family cohesion, and social supports may be related to depression among adolescents living with HIV. We used baseline data from the Suubi + Adherence study, a 5-year longitudinal randomized controlled trial among adolescents living with HIV in southwestern Uganda (n = 675; ages 10-16 years). Hierarchical logistic regression models were conducted separately among in-school adolescents and out-of-school adolescents to assess the hypothesized associations between economic and social equity, social support, and depression. About half of the participants meet the criteria for depression. Adolescents with depression were found to have fewer economic and social supports. Our findings indicate that social and economic equity [odds ratio (OR) = 0.85, 95% confidence interval (CI) 0.74, 0.99], family cohesion (OR = 0.94, 95% CI 0.91-0.96), and social support from friends (OR = 0.95, 95% CI 0.91-0.998) are associated with depression for in-school HIV infected adolescents and could be protective factors. The results of this study suggest that social and economic equity may play a protective role against depression and other poor mental health outcomes. Potential interventions for adolescents living with HIV should consider these social and familial factors as they may be protective of depression in this population.

Examining the self-reported advantages and disadvantages of socially networking about body image and eating disorders.

OBJECTIVE: The purpose of this study is to understand the self-reported advantages and disadvantages of socially networking about body image/eating disorders (EDs) and to examine the openness of these participants to online outreach and support for ED symptoms. METHOD: A cross-sectional online survey was conducted with a sample of N = 598. Eligible participants were ≥15 years old, English-speaking, and U.S. residents who endorsed posting or following thin-ideal/body-image content on social media. Quantitative measures were used to assess online peer support and online interaction preferences, and to identify ED symptoms. Deductive and inductive qualitative approaches were used to analyze open-ended items about the advantages and disadvantages of social networking about thin-ideal content on social media platforms (SMPs). RESULTS: Among those who posted about the thin-ideal on social media, 70% felt that the peer responses were positive and supportive. Participants generally favored online interaction, and a third stated that they would accept support from someone they did not know online (38%). The most common advantages noted for posting/following thin-ideal content on SMPs were motivation/encouragement to engage in a certain behavior, socializing, and information giving/seeking. The most common disadvantages mentioned for posting/following thin-ideal content on SMPs were that the content elicits negative/bad feelings, having to deal with the negative consequences/reactions of others when socially networking about this topic, and that it triggers a desire to engage in ED behaviors. DISCUSSION: With these findings, researchers, health practitioners, and social media administrators can devise ways to reduce harmful consequences of posting/following body-image/ED content on social media.

Novel REEP6 gene mutation associated with autosomal recessive retinitis pigmentosa.

PURPOSE: This study reports the ophthalmic and genetic findings of a Cameroonian patient with autosomal recessive retinitis pigmentosa (arRP) caused by a novel Receptor Expression Enhancing Protein 6 (REEP6) homozygous mutation. PATIENT AND METHODS: A 33-year-old man underwent comprehensive ophthalmic examinations, including visual acuity measurements, dilated fundus imaging, electroretinography (ERG), and spectral-domain optical coherence tomography (SD-OCT). Short-wavelength fundus autofluorescence (SW-AF) and near-infrared fundus autofluorescence (NIR-AF) were also evaluated. Whole exome sequencing (WES) was used to identify potential pathogenic variants. RESULTS: Fundus examination revealed typical RP findings with additional temporal ten micron yellow dots. SD-OCT imaging revealed cystoid macular edema and perifoveal outer retinal atrophy with centrally preserved inner segment ellipsoid zone (EZ) bands. Hyperreflective spots were seen in the inner retinal layers. On SW-AF images, a hypoautofluorescent area in the perifoveal area was observed. NIR-AF imaging revealed an irregularly shaped hyperautofluorescent ring. His visual acuity was mildly affected. ERG showed undetectable rod responses and intact cone responses. Genetic testing via WES revealed a novel homozygous mutation (c.295G>A, p.Glu99Lys) in the gene encoding REEP6, which is predicted to alter the charge in the transmembrane helix. CONCLUSIONS: This report is not only the first description of a Cameroonian patient with arRP associated with a REEP6 mutation, but also this particular genetic alteration. Substitution of p.Glu99Lys in REEP6 likely disrupts the interactions between REEP6 and the ER membrane. NIR-AF imaging may be particularly useful for assessing functional photoreceptor cells and show an "avocado" pattern of hyperautofluorescence in patients with the REEP6 mutation.

Embryonic and postnatal development of mouse olfactory tubercle.

The olfactory tubercle (OT) is located in the ventral-medial region of the brain where it receives primary input from olfactory bulb (OB) projection neurons and processes olfactory behaviors related to motivation, hedonics of smell and sexual encounters. The OT is part of the dopamine reward system that shares characteristics with the striatum. Together with the nucleus accumbens, the OT has been referred to as the "ventral striatum". However, despite its functional importance little is known about the embryonic development of the OT and the phenotypic properties of the OT cells. Here, using thymidine analogs, we establish that mouse OT neurogenesis occurs predominantly between E11-E15 in a lateral-to-medial gradient. Then, using a piggyBac multicolor technique we characterized the migratory route of OT neuroblasts from their embryonic point of origin. Following neurogenesis in the ventral lateral ganglionic eminence (vLGE), neuroblasts destined for the OT followed a dorsal-ventral pathway we named "ventral migratory course" (VMC). Upon reaching the nascent OT, neurons established a prototypical laminar distribution that was determined, in part, by the progenitor cell of origin. A phenotypic analysis of OT neuroblasts using a single-color piggyBac technique, showed that OT shared the molecular specification of striatal neurons. In addition to primary afferent input from the OB, the OT also receives a robust dopaminergic input from ventral tegmentum (Ikemoto, 2007). We used tyrosine hydroxylase (TH) expression as a proxy for dopaminergic innervation and showed that TH onset occurs at E13 and progressively increased until postnatal stages following an 'inside-out' pattern. Postnatally, we established the myelination in the OT occurring between P7 and P14, as shown with CNPase staining, and we characterized the cellular phenotypes populating the OT by immunohistochemistry. Collectively, this work provides the first detailed analysis of the developmental and maturation processes occurring in mouse OT, and demonstrates the striatal nature of the OT as part of the ventral striatum (vST).

Genetic Rescue Reverses Microglial Activation in Preclinical Models of Retinitis Pigmentosa.

Microglia cells (MGCs) play a key role in scavenging pathogens and phagocytosing cellular debris in the central nervous system and retina. Their activation, however, contributes to the progression of multiple degenerative diseases. Given the potential damage created by MGCs, it is important to better understand their mechanism of activation. Here, we explored the role of MGCs in the context of retinitis pigmentosa (RP) by using four independent preclinical mouse models. For therapeutic modeling, tamoxifen-inducible CreER was introduced to explore changes in MGCs when RP progression halted. The phenotypes of the MGCs were observed using live optical coherence tomography, live autofluorescence, and immunohistochemistry. We found that, regardless of genetic background, MGCs were activated in neurodegenerative conditions and migrated beyond the layers where they are typically found to the inner and outer segments, where degeneration was ongoing. Genetic rescue not only halted degeneration but also deactivated MGCs, regardless of whether the intervention occurred at the early, middle, or late stage of the disease. These findings suggest that halting long-term disease progression may be more successful by downregulating MGC activity while co-administering the therapeutic intervention.

Reprogramming the metabolome rescues retinal degeneration.

Metabolomics studies in the context of ophthalmology have largely focused on identifying metabolite concentrations that characterize specific retinal diseases. Studies involving mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy have shown that individuals suffering from retinal diseases exhibit metabolic profiles that markedly differ from those of control individuals, supporting the notion that metabolites may serve as easily identifiable biomarkers for specific conditions. An emerging branch of metabolomics resulting from biomarker studies, however, involves the study of retinal metabolic dysfunction as causes of degeneration. Recent publications have identified a number of metabolic processes-including but not limited to glucose and oxygen metabolism-that, when perturbed, play a role in the degeneration of photoreceptor cells. As a result, such studies have led to further research elucidating methods for prolonging photoreceptor survival in an effort to halt degeneration in its early stages. This review will explore the ways in which metabolomics has deepened our understanding of the causes of retinal degeneration and discuss how metabolomics can be used to prevent retinal degeneration from progressing to its later disease stages.

Correlation between B-scan optical coherence tomography, en face thickness map ring and hyperautofluorescent ring in retinitis pigmentosa patients.

OBJECTIVE: To evaluate and compare the B-scan OCT loss of ellipsoid zone, OCT en face thickness map constriction, and hyperautofluorescent ring constriction in RP patients. METHODS: Retrospective case series study. Forty-eight eyes of 24 RP patients with a parafoveal hyperautofluorescent ring were studied. The diagnosis of RP was established by the presence of rod response impairment and a prevalent decrease of scotopic over photopic responses on electroretinography. The FAF and spectral-domain optical coherence tomography (SD-OCT) images were obtained from 24 patients with RP. The measurements of the EZ line width on B-scan OCT, hyperautofluorescent ring area on FAF, and hyperautofluorescent ring area on en face thickness map were performed by two independent graders. The measurements of these three parameters were correlated. RESULTS: The mean age of study patients was 46 years old (sd = 19). The external and internal FAF rings involving the fovea were identified in all study eyes. The area of the thickness ring decreased at an average rate of 0.5 (sd 0.4) mm2 per year (P < 0.001). The average rate of EZ-line constriction was estimated to be 123 (sd 63) µm per year (P < 0.001). The hyperautofluorescent ring area decreased at an average rate of 0.9 (sd 0.98) mm2 per year (P < 0.001). The strongest correlation was observed between hyperautofluorescent ring area and EZ-line width (r = 0.78). CONCLUSIONS: We observed that the hyperautofluorescent ring area exhibits a faster progression rate than the area of the thickness ring. In addition, we found that the EZ-line width had a high positive correlation with the hyperautofluorescent ring area and a moderate positive correlation with area of the thickness ring.

Fundus autofluorescence and ellipsoid zone (EZ) line width can be an outcome measurement in RHO-associated autosomal dominant retinitis pigmentosa.

PURPOSE: To evaluate the progression of retinitis pigmentosa (RP) due to mutations in rhodopsin (RHO) by measuring the short-wavelength autofluorescence (SW-AF) increased autofluorescence ring and ellipsoid zone (EZ)-line width. METHODS: Fundus autofluorescence (FAF) and spectral domain optical coherence tomography (SD-OCT) images were obtained from 10 patients with autosomal dominant RP due to mutations in the RHO gene. Measurements of ring area on FAF images, as well as the EZ line width on SD-OCT images and horizontal, vertical diameter, were performed by two independent masked graders. RESULTS: The ring area decreased by a rate of 0.6 ± 0.2 mm2 per year. We observed that the EZ line width decreased by an average of 152 ± 37 µm per year, while the horizontal and vertical diameters decreased by 106 ± 35 µm and 125 ± 29 µm per year, respectively. Progression rates were similar between eyes. CONCLUSIONS: We observed SW-AF ring constriction and a progressive loss of EZ line width over time.

Clustered Regularly Interspaced Short Palindromic Repeats-Based Genome Surgery for the Treatment of Autosomal Dominant Retinitis Pigmentosa.

PURPOSE: To develop a universal gene therapy to overcome the genetic heterogeneity in retinitis pigmentosa (RP) resulting from mutations in rhodopsin (RHO). DESIGN: Experimental study for a combination gene therapy that uses both gene ablation and gene replacement. PARTICIPANTS: This study included 2 kinds of human RHO mutation knock-in mouse models: RhoP23H and RhoD190N. In total, 23 RhoP23H/P23H, 43 RhoP23H/+, and 31 RhoD190N/+ mice were used for analysis. METHODS: This study involved gene therapy using dual adeno-associated viruses (AAVs) that (1) destroy expression of the endogenous Rho gene in a mutation-independent manner via an improved clustered regularly interspaced short palindromic repeats-based gene deletion and (2) enable expression of wild-type protein via exogenous cDNA. MAIN OUTCOME MEASURES: Electroretinographic and histologic analysis. RESULTS: The thickness of the outer nuclear layer (ONL) after the subretinal injection of combination ablate-and-replace gene therapy was approximately 17% to 36% more than the ONL thickness resulting from gene replacement-only therapy at 3 months after AAV injection. Furthermore, electroretinography results demonstrated that the a and b waves of both RhoP23H and RhoD190N disease models were preserved more significantly using ablate-and-replace gene therapy (P < 0.001), but not by gene replacement monotherapy. CONCLUSIONS: As a proof of concept, our results suggest that the ablate-and-replace strategy can ameliorate disease progression as measured by photoreceptor structure and function for both of the human mutation knock-in models. These results demonstrate the potency of the ablate-and-replace strategy to treat RP caused by different Rho mutations. Furthermore, because ablate-and-replace treatment is mutation independent, this strategy may be used to treat a wide array of dominant diseases in ophthalmology and other fields. Clinical trials using ablate-and-replace gene therapy would allow researchers to determine if this strategy provides any benefits for patients with diseases of interest.

CRISPR/Cas9 genome surgery for retinal diseases.

Retinal diseases that impair vision can impose heavy physical and emotional burdens on patients' lives. Currently, clustered regularly interspaced short palindromic repeats (CRISPR) is a prevalent gene-editing tool that can be harnessed to generate disease model organisms for specific retinal diseases, which are useful for elucidating pathophysiology and revealing important links between genetic mutations and phenotypic defects. These retinal disease models are fundamental for testing various therapies and are indispensible for potential future clinical trials. CRISPR-mediated procedures involving CRISPR-associated protein 9 (Cas9) may also be used to edit genome sequences and correct mutations. Thus, if used for future therapies, CRISPR/Cas9 genome surgery could eliminate the need for patients with retinal diseases to undergo repetitive procedures such as drug injections. In this review, we will provide an overview of CRISPR/Cas9, discuss the different types of Cas9, and compare Cas9 to other endonucleases. Furthermore, we will explore the many ways in which researchers are currently utilizing this versatile tool, as CRISPR/Cas9 may have far-reaching effects in the treatment of retinal diseases.