Intravitreal injection of a rationally designed AAV capsid library in non-human primate identifies variants with enhanced retinal transduction and neutralizing antibody evasion.

Adeno-associated virus (AAV) continues to be the gold standard vector for therapeutic gene delivery and has proven especially useful for treating ocular disease. Intravitreal injection (IVtI) is a promising delivery route because it increases accessibility of gene therapies to larger patient populations. However, data from clinical and non-human primate (NHP) studies utilizing currently available capsids indicate that anatomical barriers to AAV and pre-existing neutralizing antibodies can restrict gene expression to levels that are "sub-therapeutic" in a substantial proportion of patients. Here, we performed a combination of directed evolution in NHPs of an AAV2-based capsid library with simultaneous mutations across six surface-exposed variable regions and rational design to identify novel capsid variants with improved retinal transduction following IVtI. Following two rounds of screening in NHP, enriched variants were characterized in intravitreally injected mice and NHPs and shown to have increased transduction relative to AAV2. Lead capsid variant, P2-V1, demonstrated an increased ability to evade neutralizing antibodies in human vitreous samples relative to AAV2 and AAV2.7m8. Taken together, this study further contributed to our understanding of the selective pressures associated with retinal transduction via the vitreous and identified promising novel AAV capsid variants for clinical consideration.

Preclinical studies in support of phase I/II clinical trials to treat GUCY2D-associated Leber congenital amaurosis.

Mutations in GUCY2D are associated with severe early-onset retinal dystrophy, Leber congenital amaurosis type 1 (LCA1), a leading cause of blindness in children. Despite a high degree of visual disturbance stemming from photoreceptor dysfunction, patients with LCA1 largely retain normal photoreceptor structure, suggesting that they are good candidates for gene replacement therapy. The purpose of this study was to conduct the preclinical and IND-enabling experiments required to support clinical application of AAV5-hGRK1-GUCY2D in patients harboring biallelic recessive mutations in GUCY2D. Preclinical studies were conducted in mice to evaluate the effect of vector manufacturing platforms and transgene species on the therapeutic response. Dose-ranging studies were conducted in cynomolgus monkeys to establish the minimum dose required for efficient photoreceptor transduction. Good laboratory practice (GLP) studies evaluated systemic biodistribution in rats and toxicology in non-human primates (NHPs). These results expanded our knowledge of dose response for an AAV5-vectored transgene under control of the human rhodopsin kinase (hGRK1) promoter in NHPs with respect to photoreceptor transduction and safety and, in combination with the rat biodistribution and mouse efficacy studies, informed the design of a first-in-human clinical study in patients with LCA1.

A Genomically and Clinically Annotated Patient-Derived Xenograft Resource for Preclinical Research in Non-Small Cell Lung Cancer.

Patient-derived xenograft (PDX) models are an effective preclinical in vivo platform for testing the efficacy of novel drugs and drug combinations for cancer therapeutics. Here we describe a repository of 79 genomically and clinically annotated lung cancer PDXs available from The Jackson Laboratory that have been extensively characterized for histopathologic features, mutational profiles, gene expression, and copy-number aberrations. Most of the PDXs are models of non-small cell lung cancer (NSCLC), including 37 lung adenocarcinoma (LUAD) and 33 lung squamous cell carcinoma (LUSC) models. Other lung cancer models in the repository include four small cell carcinomas, two large cell neuroendocrine carcinomas, two adenosquamous carcinomas, and one pleomorphic carcinoma. Models with both de novo and acquired resistance to targeted therapies with tyrosine kinase inhibitors are available in the collection. The genomic profiles of the LUAD and LUSC PDX models are consistent with those observed in patient tumors from The Cancer Genome Atlas and previously characterized gene expression-based molecular subtypes. Clinically relevant mutations identified in the original patient tumors were confirmed in engrafted PDX tumors. Treatment studies performed in a subset of the models recapitulated the responses expected on the basis of the observed genomic profiles. These models therefore serve as a valuable preclinical platform for translational cancer research. SIGNIFICANCE: Patient-derived xenografts of lung cancer retain key features observed in the originating patient tumors and show expected responses to treatment with standard-of-care agents, providing experimentally tractable and reproducible models for preclinical investigations.

Identification of Arhgef12 and Prkci as genetic modifiers of retinal dysplasia in the Crb1rd8 mouse model.

Mutations in the apicobasal polarity gene CRB1 lead to diverse retinal diseases, such as Leber congenital amaurosis, cone-rod dystrophy, retinitis pigmentosa (with and without Coats-like vasculopathy), foveal retinoschisis, macular dystrophy, and pigmented paravenous chorioretinal atrophy. Limited correlation between disease phenotypes and CRB1 alleles, and evidence that patients sharing the same alleles often present with different disease features, suggest that genetic modifiers contribute to clinical variation. Similarly, the retinal phenotype of mice bearing the Crb1 retinal degeneration 8 (rd8) allele varies with genetic background. Here, we initiated a sensitized chemical mutagenesis screen in B6.Cg-Crb1rd8/Pjn, a strain with a mild clinical presentation, to identify genetic modifiers that cause a more severe disease phenotype. Two models from this screen, Tvrm266 and Tvrm323, exhibited increased retinal dysplasia. Genetic mapping with high-throughput exome and candidate-gene sequencing identified causative mutations in Arhgef12 and Prkci, respectively. Epistasis analysis of both strains indicated that the increased dysplastic phenotype required homozygosity of the Crb1rd8 allele. Retinal dysplastic lesions in Tvrm266 mice were smaller and caused less photoreceptor degeneration than those in Tvrm323 mice, which developed an early, large diffuse lesion phenotype. At one month of age, Müller glia and microglia mislocalization at dysplastic lesions in both modifier strains was similar to that in B6.Cg-Crb1rd8/Pjn mice but photoreceptor cell mislocalization was more extensive. External limiting membrane disruption was comparable in Tvrm266 and B6.Cg-Crb1rd8/Pjn mice but milder in Tvrm323 mice. Immunohistological analysis of mice at postnatal day 0 indicated a normal distribution of mitotic cells in Tvrm266 and Tvrm323 mice, suggesting normal early development. Aberrant electroretinography responses were observed in both models but functional decline was significant only in Tvrm323 mice. These results identify Arhgef12 and Prkci as modifier genes that differentially shape Crb1-associated retinal disease, which may be relevant to understanding clinical variability and underlying disease mechanisms in humans.

Metabolic Adaptation of Macrophages as Mechanism of Defense against Crystalline Silica.

Silicosis is a lethal pneumoconiosis for which no therapy is available. Silicosis is a global threat, and more than 2.2 million people per year are exposed to silica in the United States. The initial response to silica is mediated by innate immunity. Phagocytosis of silica particles by macrophages is followed by recruitment of mitochondria to phagosomes, generation of mitochondrial reactive oxygen species, and cytokine (IL-1ß, TNF-α, IFN-ß) release. In contrast with LPS, the metabolic remodeling of silica-exposed macrophages is unclear. This study contrasts mitochondrial and metabolic alterations induced by LPS and silica on macrophages and correlates them with macrophage viability and cytokine production, which are central to the pathogenesis of silicosis. Using high-resolution respirometer and liquid chromatography-high-resolution mass spectrometry, we determined the effects of silica and LPS on mitochondrial respiration and determined changes in central carbon metabolism of murine macrophage cell lines RAW 264.7 and IC-21. We show that silica induces metabolic reprogramming of macrophages. Silica, as well as LPS, enhances glucose uptake and increases aerobic glycolysis in macrophages. In contrast with LPS, silica affects mitochondria respiration, reducing complex I and enhancing complex II activity, to sustain cell viability. These mitochondrial alterations are associated in silica, but not in LPS-exposed macrophages, with reductions of tricarboxylic acid cycle intermediates, including succinate, itaconate, glutamate, and glutamine. Furthermore, in contrast with LPS, these silica-induced metabolic adaptations do not correlate with IL-1ß or TNF-α production, but with the suppressed release of IFN-ß. Our data highlight the importance of complex II activity and tricarboxylic acid cycle remodeling to macrophage survival and cytokine-mediated inflammation in silicosis.

Effects of Altering HSPG Binding and Capsid Hydrophilicity on Retinal Transduction by AAV.

Adeno-associated viruses (AAVs) have recently emerged as the leading vector for retinal gene therapy. However, AAV vectors which are capable of achieving clinically relevant levels of transgene expression and widespread retinal transduction are still an unmet need. Using rationally designed AAV2-based capsid variants, we investigate the role of capsid hydrophilicity and hydrophobicity as it relates to retinal transduction. We show that hydrophilic, single amino acid (aa) mutations (V387R, W502H, E530K, L583R) in AAV2 negatively impact retinal transduction when heparan sulfate proteoglycan (HSPG) binding remains intact. Conversely, addition of hydrophobic point mutations to an HSPG binding deficient capsid (AAV2ΔHS) lead to increased retinal transduction in both mouse and macaque. Our top performing vector, AAV2(4pMut)ΔHS, achieved robust rod and cone photoreceptor (PR) transduction in macaque, especially in the fovea, and demonstrates the ability to spread laterally beyond the borders of the subretinal injection (SRI) bleb. This study both evaluates biophysical properties of AAV capsids that influence retinal transduction, and assesses the transduction and tropism of a novel capsid variant in a clinically relevant animal model.ImportanceRationally guided engineering of AAV capsids aims to create new generations of vectors with enhanced potential for human gene therapy. By applying rational design principles to AAV2-based capsids, we evaluated the influence of hydrophilic and hydrophobic amino acid (aa) mutations on retinal transduction as it relates to vector administration route. Through this approach we identified a largely deleterious relationship between hydrophilic aa mutations and canonical HSPG binding by AAV2-based capsids. Conversely, the inclusion of hydrophobic aa substitutions on a HSPG binding deficient capsid (AAV2ΔHS), generated a vector capable of robust rod and cone photoreceptor (PR) transduction. This vector AAV2(4pMut)ΔHS also demonstrates a remarkable ability to spread laterally beyond the initial subretinal injection (SRI) bleb, making it an ideal candidate for the treatment of retinal diseases which require a large area of transduction.

Not all wavelengths are created equal: disinfection of SARS-CoV-2 using UVC radiation is wavelength-dependent.

SARS-CoV-2 is mostly transmitted through close contact with infected people by infected aerosols and fomites. Ultraviolet subtype C (UVC) lamps and light-emitting diodes can be used to disrupt the transmission chain by disinfecting fomites, thus managing the disease outbreak progression. Here, we assess the ultraviolet wavelengths that are most effective in inactivation of SARS-CoV-2 on fomites. Variations in UVC wavelengths impact the dose required for disinfection of SARS-CoV-2 and alter how rapidly and effectively disruption of the virus transmission chain can be achieved. This study reveals that shorter wavelengths (254-268 nm) take a maximum of 6.25 mJ/cm2 over 5 s to obtain a target SARS-CoV-2 reduction of 99.9%. Longer wavelengths, like 280 nm, take longer irradiation time and higher dose to inactivate SARS-CoV-2. These observations emphasize that SARS-CoV-2 inactivation is wavelength-dependent.

Mechanisms of Plutonium Redox Reactions in Nitric Acid Solutions.

Plutonium (Pu) exhibits a complex redox behavior in aqueous solutions. This is due to the ability of the element to adapt a wide range of oxidation states typically from +3 to +6 and the tendency for dynamic interconversion between the oxidation states that primarily depend upon acid concentration and presence of coordinating ligands. This work interrogates the Pu redox behavior in aqueous nitric acid via a combination of voltammetry and in situ vis-NIR spectroelectrochemistry under controlled potentials to map the interconversion between the various Pu oxidation states. The NIR-spectroelectrochemistry studies used to complement the visible spectroscopy bring a new and more complete perspective into the plutonium redox transformations. This allows elucidation of the mechanisms of the involved redox reactions facilitating an in-depth understanding of the relative stability of the Pu oxidation states as a function of redox potentials and nitric acid concentrations. It is observed that oxidation of Pu(III) results in generation of Pu(IV) and Pu(VI) (the latter as PuO22+), bypassing the Pu(V) oxidation state. Further, with increasing acid concentrations, the formation of the Pu(VI) species progressively decreases so that the dynamic equilibrium between the Pu(III) and Pu(IV) oxidation states dominates. These findings have significant implications for developing separation processes for used nuclear fuel reprocessing and treatment.

Novel AAV44.9-Based Vectors Display Exceptional Characteristics for Retinal Gene Therapy.

The majority of inherited retinal diseases (IRDs) are caused by mutations in genes expressed in photoreceptors (PRs). The ideal vector to address these conditions is one that transduces PRs in large areas of retina with the smallest volume/lowest titer possible, and efficiently transduces foveal cones, the cells responsible for acute, daylight vision that are often the only remaining area of functional retina in IRDs. The purpose of our study was to evaluate the retinal tropism and potency of a novel capsid, AAV44.9, and rationally designed derivatives thereof. We found that AAV44.9 and AAV44.9(E531D) transduced retinas of subretinally injected (SRI) mice with higher efficiency than did benchmark AAV5- and AAV8-based vectors. In macaques, highly efficient cone and rod transduction was observed following submacular and peripheral SRI. AAV44.9- and AAV44.9(E531D)-mediated GFP fluorescence extended laterally well beyond SRI bleb margins. Notably, extrafoveal injection (i.e., fovea not detached during surgery) led to transduction of up to 98% of foveal cones. AAV44.9(E531D) efficiently transduced parafoveal and perifoveal cones, whereas AAV44.9 did not. AAV44.9(E531D) was also capable of restoring retinal function to a mouse model of IRD. These novel capsids will be useful for addressing IRDs that would benefit from an expansive treatment area.

Somatic Gene Editing of GUCY2D by AAV-CRISPR/Cas9 Alters Retinal Structure and Function in Mouse and Macaque.

Mutations in GUCY2D, the gene encoding retinal guanylate cyclase-1 (retGC1), are the leading cause of autosomal dominant cone-rod dystrophy (CORD6). Significant progress toward clinical application of gene replacement therapy for Leber congenital amaurosis (LCA) due to recessive mutations in GUCY2D (LCA1) has been made, but a different approach is needed to treat CORD6 where gain of function mutations cause dysfunction and dystrophy. The CRISPR/Cas9 gene editing system efficiently disrupts genes at desired loci, enabling complete gene knockout or homology directed repair. Here, adeno-associated virus (AAV)-delivered CRISPR/Cas9 was used specifically to edit/disrupt this gene's early coding sequence in mouse and macaque photoreceptors in vivo, thereby knocking out retGC1 expression and demonstrably altering retinal function and structure. Neither preexisting nor induced Cas9-specific T-cell responses resulted in ocular inflammation in macaques, nor did it limit GUCY2D editing. The results show, for the first time, the ability to perform somatic gene editing in primates using AAV-CRISPR/Cas9 and demonstrate the viability this approach for treating inherited retinal diseases in general and CORD6 in particular.

Immunogenicity and Safety of the HZ/su Adjuvanted Herpes Zoster Subunit Vaccine in Adults Previously Vaccinated With a Live Attenuated Herpes Zoster Vaccine.

Background: Protection against herpes zoster (HZ) induced by the live attenuated zoster vaccine Zostavax (ZVL) wanes within 3-7 years. Revaccination may renew protection. We assessed whether (re)vaccination with the adjuvanted HZ subunit vaccine candidate (HZ/su) induced comparable immune responses in previous ZVL recipients and ZVL-naive individuals (HZ-NonVac). Methods: In an open-label, multicenter study, adults ≥65 years of age, vaccinated with ZVL ≥5 years previously (HZ-PreVac), were matched to ZVL-naive adults (HZ-NonVac). Participants received 2 doses of HZ/su 2 months apart. The primary objective of noninferiority of the humoral immune response 1 month post-dose 2 was considered demonstrated if the upper limit of the 95% confidence interval (CI) of the adjusted anti-glycoprotein E geometric mean concentration (GMC) ratio of HZ-NonVac over HZ-PreVac was <1.5. HZ/su cellular immunogenicity, reactogenicity, and safety were also assessed. Results: In 430 participants, humoral immune response to HZ/su was noninferior in HZ-PreVac compared with HZ-NonVac (adjusted GMC ratio, 1.04 [95% CI, .92-1.17]). Cellular immunogenicity, reactogenicity, and safety appeared to be comparable between groups. HZ/su was well-tolerated, with no safety concerns raised within 1 month post-dose 2. Conclusions: HZ/su induces a strong immune response irrespective of prior vaccination with ZVL, and may be an attractive option to revaccinate prior ZVL recipients. Clinical Trials Registration: NCT02581410.

Optimization of Retinal Gene Therapy for X-Linked Retinitis Pigmentosa Due to RPGR Mutations.

X-linked retinitis pigmentosa (XLRP) caused by mutations in the RPGR gene is an early onset and severe cause of blindness. Successful proof-of-concept studies in a canine model have recently shown that development of a corrective gene therapy for RPGR-XLRP may now be an attainable goal. In preparation for a future clinical trial, we have here optimized the therapeutic AAV vector construct by showing that GRK1 (rather than IRBP) is a more efficient promoter for targeting gene expression to both rods and cones in non-human primates. Two transgenes were used in RPGR mutant (XLPRA2) dogs under the control of the GRK1 promoter. First was the previously developed stabilized human RPGR (hRPGRstb). Second was a new full-length stabilized and codon-optimized human RPGR (hRPGRco). Long-term (>2 years) studies with an AAV2/5 vector carrying hRPGRstb under control of the GRK1 promoter showed rescue of rods and cones from degeneration and retention of vision. Shorter term (3 months) studies demonstrated comparable preservation of photoreceptors in canine eyes treated with an AAV2/5 vector carrying either transgene under the control of the GRK1 promoter. These results provide the critical molecular components (GRK1 promoter, hRPGRco transgene) to now construct a therapeutic viral vector optimized for RPGR-XLRP patients.

Comprehensive genomic and phenotypic characterization of germline FH deletion in hereditary leiomyomatosis and renal cell carcinoma.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a familial cancer syndrome associated with the development of cutaneous and uterine leiomyomas, and an aggressive form of type 2 papillary kidney cancer. HLRCC is characterized by germline mutation of the FH gene. This study evaluated the prevalence and clinical phenotype of FH deletions in HLRCC patients. Patients with phenotypic manifestations consistent with HLRCC who lacked detectable germline FH intragenic mutations were investigated for FH deletion. A series of 28 patients from 13 families were evaluated using a combination of a comparative genomic hybridization (CGH) array and/or CLIA-approved FH deletion/duplication analyses. Thirteen distinct germline deletions were identified in the 13 UOB families, including 11 complete FH gene deletions and 2 partial FH gene deletions. The size of eight evaluated complete FH deletions varied from ∼4.74 Mb to 249 kb, with all deletions resulting in additional gene losses. Two partial FH gene deletions were identified, with one resulting in loss of exon 1 and the upstream region of the FH gene only. Kidney cancer was diagnosed in 9 (32%) of 28 patients and 7 (54%) of 13 families possessing either complete or partial FH deletions. Cutaneous and uterine leiomyomas were observed at similar rates to those in FH point mutation families. Complete or partial FH gene alterations in HLRCC families are associated with all of the canonical HLRCC manifestations, including type 2 papillary kidney cancer and should be screened for in any patient at-risk for this disorder.

Highly Efficient Delivery of Adeno-Associated Viral Vectors to the Primate Retina.

Adeno-associated virus (AAV) has emerged as the preferred vector for targeting gene expression to the retina. Subretinally injected AAV can efficiently transduce retinal pigment epithelium and photoreceptors in primate retina. Inner and middle primate retina can be transduced by intravitreally delivered AAV, but with low efficiency. This is due to dilution of vector, potential neutralization of capsid because it is not confined to the immune-privileged retinal compartment, and the presence of the inner limiting membrane (ILM), a barrier separating the vitreous from the neural retina. We here describe a novel "subILM" injection method that addresses all three issues. Specifically, vector is placed in a surgically induced, hydrodissected space between the ILM and neural retina. In an initial experiment, we injected viscoelastic (Healon(®)), a substance we confirmed was biocompatible with AAV, to create a subILM bleb and subsequently injected AAV2-GFP into the bleb after irrigation with basic salt solution. For later experiments, we used a Healon-AAV mixture to place single, subILM injections. In all cases, subILM delivery of AAV was well tolerated-no inflammation or gross structural changes were observed by ophthalmological examination or optical coherence tomography. In-life fluorescence imaging revealed profound transgene expression within the area of the subILM injection bleb that persisted for the study duration. Uniform and extensive transduction of retinal ganglion cells (RGCs) was achieved in the areas beneath the subILM bleb. Transduction of Müller glia, ON bipolar cells, and photoreceptors was also observed. Robust central labeling from green fluorescent protein-expressing RGCs confirmed their continued survival, and was observed in the lateral geniculate nucleus, the superior colliculus, and the pretectum. Our results confirm that the ILM is a major barrier to transduction by AAV in primate retina and that, when it is circumvented, the efficiency and depth to which AAV2 promotes transduction of multiple retinal cell classes are greatly enhanced.

Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-Associated Virus Vectors.

UNLABELLED: Adeno-associated viruses (AAVs) currently are being developed to efficiently transduce the retina following noninvasive, intravitreal (Ivt) injection. However, a major barrier encountered by intravitreally delivered AAVs is the inner limiting membrane (ILM), a basement membrane rich in heparan sulfate (HS) proteoglycan. The goal of this study was to determine the impact of HS binding on retinal transduction by Ivt-delivered AAVs. The heparin affinities of AAV2-based tyrosine-to-phenylalanine (Y-F) and threonine-to-valine (T-V) capsid mutants, designed to avoid proteasomal degradation during cellular trafficking, were established. In addition, the impact of grafting HS binding residues onto AAV1, AAV5, and AAV8(Y733F) as well as ablation of HS binding by AAV2-based vectors on retinal transduction was investigated. Finally, the potential relationship between thermal stability of AAV2-based capsids and Ivt-mediated transduction was explored. The results show that the Y-F and T-V AAV2 capsid mutants bind heparin but with slightly reduced affinity relative to that of AAV2. The grafting of HS binding increased Ivt transduction by AAV1 but not by AAV5 or AAV8(Y733F). The substitution of any canonical HS binding residues ablated Ivt-mediated transduction by AAV2-based vectors. However, these same HS variant vectors displayed efficient retinal transduction when delivered subretinally. Notably, a variant devoid of canonical HS binding residues, AAV2(4pMut)ΔHS, was remarkably efficient at transducing photoreceptors. The disparate AAV phenotypes indicate that HS binding, while critical for AAV2-based vectors, is not the sole determinant for transduction via the Ivt route. Finally, Y-F and T-V mutations alter capsid stability, with a potential relationship existing between stability and improvements in retinal transduction by Ivt injection. IMPORTANCE: AAV has emerged as the vector of choice for gene delivery to the retina, with attention focused on developing vectors that can mediate transduction following noninvasive, intravitreal injection. HS binding has been postulated to play a role in intravitreally mediated transduction of retina. Our evaluation of the HS binding of AAV2-based variants and other AAV serotype vectors and the correlation of this property with transduction points to HS affinity as a factor controlling retinal transduction following Ivt delivery. However, HS binding is not the only requirement for improved Ivt-mediated transduction. We show that AAV2-based vectors lacking heparin binding transduce retina by subretinal injection and display a remarkable ability to transduce photoreceptors, indicating that other receptors are involved in this phenotype.

Cone-Specific Promoters for Gene Therapy of Achromatopsia and Other Retinal Diseases.

Adeno-associated viral (AAV) vectors containing cone-specific promoters have rescued cone photoreceptor function in mouse and dog models of achromatopsia, but cone-specific promoters have not been optimized for use in primates. Using AAV vectors administered by subretinal injection, we evaluated a series of promoters based on the human L-opsin promoter, or a chimeric human cone transducin promoter, for their ability to drive gene expression of green fluorescent protein (GFP) in mice and nonhuman primates. Each of these promoters directed high-level GFP expression in mouse photoreceptors. In primates, subretinal injection of an AAV-GFP vector containing a 1.7-kb L-opsin promoter (PR1.7) achieved strong and specific GFP expression in all cone photoreceptors and was more efficient than a vector containing the 2.1-kb L-opsin promoter that was used in AAV vectors that rescued cone function in mouse and dog models of achromatopsia. A chimeric cone transducin promoter that directed strong GFP expression in mouse and dog cone photoreceptors was unable to drive GFP expression in primate cones. An AAV vector expressing a human CNGB3 gene driven by the PR1.7 promoter rescued cone function in the mouse model of achromatopsia. These results have informed the design of an AAV vector for treatment of patients with achromatopsia.

Comparing Cost-Effectiveness of HIV Testing Strategies: Targeted and Routine Testing in Washington, DC.

BACKGROUND: Routine HIV testing is an essential approach to identifying undiagnosed infections, linking people to care and treatment, and preventing new infections. In Washington, DC, where HIV prevalence is 2.4%, a combination of routine and targeted testing approaches has been implemented since 2006. METHODS: We sought to evaluate the cost effectiveness of the District of Columbia (DC) Department of Health's routine and targeted HIV testing implementation strategies. We collected HIV testing data from 3 types of DC Department of Health-funded testing sites (clinics, hospitals, and community-based organizations); collected testing and labor costs; and calculated effectiveness measures including cost per new diagnosis and cost per averted transmission. RESULTS: Compared to routine testing, targeted testing resulted in higher positivity rates (1.33% vs. 0.44%). Routine testing averted 34.30 transmissions per year compared to targeted testing at 17.78. The cost per new diagnosis was lower for targeted testing ($2,467 vs. $7,753 per new diagnosis) as was the cost per transmission averted ($33,160 vs. $104,205). When stratified by testing site, both testing approaches were most cost effective in averting new transmissions when conducted by community based organizations ($25,037 routine; $33,123 targeted) compared to hospitals or clinics. CONCLUSIONS: While routine testing identified more newly diagnosed infections and averted more infections than targeted testing, targeted testing is more cost effective per diagnosis and per transmission averted overall. Given the high HIV prevalence in DC, the DC Department of Health's implementation strategy should continue to encourage routine testing implementation with emphasis on a combined testing strategy among community-based organizations.

Systemic Vascular Transduction by Capsid Mutant Adeno-Associated Virus After Intravenous Injection.

The ability to effectively deliver genetic material to vascular endothelial cells remains one of the greatest unmet challenges facing the development of gene therapies to prevent diseases with underlying vascular etiology, such as diabetes, atherosclerosis, and age-related macular degeneration. Herein, we assess the effectiveness of an rAAV2-based capsid mutant vector (Y272F, Y444F, Y500F, Y730F, T491V; termed QuadYF+TV) with strong endothelial cell tropism at transducing the vasculature after systemic administration. Intravenous injection of QuadYF+TV resulted in widespread transduction throughout the vasculature of several major organ systems, as assessed by in vivo bioluminescence imaging and postmortem histology. Robust transduction of lung tissue was observed in QuadYF+TV-injected mice, indicating a role for intravenous gene delivery in the treatment of chronic diseases presenting with pulmonary complications, such as α1-antitrypsin deficiency. The QuadYF+TV vector cross-reacted strongly with AAV2 neutralizing antibodies, however, indicating that a targeted delivery strategy may be required to maximize clinical translatability.

Effects of Age and Estrogen on Skeletal Gene Expression in Humans as Assessed by RNA Sequencing.

UNLABELLED: Precise delineation of the specific genes and pathways altered with aging and estrogen (E) therapy may lead to new skeletal biomarkers and the development of novel bone therapeutics. Previous human bone studies, however, have been limited by only examining pre-specified genes and pathways. High-throughput RNA sequencing (RNAseq), on the other hand, offers an unbiased approach to examine the entire transcriptome. Here we present an RNAseq analysis of human bone samples, obtained from iliac crest needle biopsies, to yield the first in vivo interrogation of all genes and pathways that may be altered in bone with aging and E therapy in humans. 58 healthy women were studied, including 19 young women (mean age ± SD, 30.3 ± 5.4 years), 19 old women (73.1 ± 6.6 years), and 20 old women treated with 3 weeks of E therapy (70.5 ± 5.2 years). Using generally accepted criteria (false discovery rate [q] < 0.10), aging altered a total of 678 genes and 12 pathways, including a subset known to regulate bone metabolism (e.g., Notch). Interestingly, the LEF1 transcription factor, which is a classical downstream target of the Wnt/ß-catenin signaling pathway, was significantly downregulated in the bones from the old versus young women; consistent with this, LEF1 binding sites were significantly enriched in the promoter regions of the differentially expressed genes in the old versus young women, suggesting that aging was associated with alterations in Wnt signaling in bone. Further, of the 21 unique genes altered in bone by E therapy, the expression of INHBB (encoding for the inhibin, beta B polypeptide), which decreased with aging (by 0.6-fold), was restored to young adult levels in response to E therapy. In conclusion, our data demonstrate that aging alters a substantial portion of the skeletal transcriptome, whereas E therapy appears to have significant, albeit less wide-ranging effects. These data provide a valuable resource for the potential identification of novel biomarkers associated with age-related bone loss and also highlight potential pathways that could be targeted to treat osteoporosis. TRIAL REGISTRATION: ClinicalTrials.gov NCT02349113.

Gene Therapy Fully Restores Vision to the All-Cone Nrl(-/-) Gucy2e(-/-) Mouse Model of Leber Congenital Amaurosis-1.

Mutations in GUCY2D are the cause of Leber congenital amaurosis type 1 (LCA1). GUCY2D encodes retinal guanylate cyclase-1 (retGC1), a protein expressed exclusively in outer segments of photoreceptors and essential for timely recovery from photoexcitation. Recent clinical data show that, despite a high degree of visual disturbance stemming from a loss of cone function, LCA1 patients retain normal photoreceptor architecture, except for foveal cone outer segment abnormalities and, in some patients, foveal cone loss. These results point to the cone-rich central retina as a target for GUCY2D replacement. LCA1 gene replacement studies thus far have been conducted in rod-dominant models (mouse) or with vectors and organisms lacking clinical translatability. Here we investigate gene replacement in the Nrl(-/-) Gucy2e(-/-) mouse, an all-cone model deficient in retGC1. We show that AAV-retGC1 treatment fully restores cone function, cone-mediated visual behavior, and guanylate cyclase activity, and preserves cones in treated Nrl(-/-) Gucy2e(-/-) mice over the long-term. A novel finding was that retinal function could be restored to levels above that in Nrl(-/-) controls, contrasting results in other models of retGC1 deficiency. We attribute this to increased cyclase activity in treated Nrl(-/-) Gucy2e(-/-) mice relative to Nrl(-/-) controls. Thus, Nrl(-/-) Gucy2e(-/-) mice possess an expanded dynamic range in ERG response to gene replacement relative to other models. Lastly, we show that a candidate clinical vector, AAV5-GRK1-GUCY2D, when delivered to adult Nrl(-/-) Gucy2e(-/-) mice, restores retinal function that persists for at least 6 months. Our results provide strong support for clinical application of a gene therapy targeted to the cone-rich, central retina of LCA1 patients.