Mammalian cones respond to light by closing a cGMP-gated channel via a cascade that includes a heterotrimeric G-protein, cone transducin, comprising G␣t2, G␤3 and G␥t2 subunits. The function of G␤␥ in this cascade has not been examined. Here, we investigate the role of G␤3 by assessing cone structure and function in G␤3-null mouse (Gnb3 Ϫ/Ϫ ). We found that G␤3 is required for the normal expression of its partners, because in the Gnb3 Ϫ/Ϫ cone outer segments, the levels of G␣t2 and G␥t2 are reduced by fourfold to sixfold, whereas other components of the cascade remain unaltered. Surprisingly, Gnb3Ϫ/Ϫ cones produce stable responses with normal kinetics and saturating response amplitudes similar to that of the wild-type, suggesting that cone phototransduction can function efficiently without a G␤ subunit. However, light sensitivity was reduced by approximately fourfold in the knock-out cones. Because the reduction in sensitivity was similar in magnitude to the reduction in G␣t2 level in the cone outer segment, we conclude that activation of G␣t2 in Gnb3 Ϫ/Ϫ cones proceeds at a rate approximately proportional to its outer segment concentration, and that activation of phosphodiesterase and downstream cascade components is normal. These results suggest that the main role of G␤3 in cones is to establish optimal levels of transducin heteromer in the outer segment, thereby indirectly contributing to robust response properties.

Purpose Gene therapy (GT) has offered immense hope to individuals who are visually impaired due to RPE65 mutations. While GT has shown great success in clinical trials enrolling these individuals, evidence for stability and durability of this treatment over time is still unknown. Here we explore the value of functional magnetic resonance imaging (fMRI) as an objective measure to independently assess the longevity of retinal GT. Design Individuals with RPE65 mutations, who underwent GT in their worse-seeing eye in a Phase I clinical trial, received a second subretinal injection in their contralateral eye in a follow-on clinical trial. fMRI was longitudinally performed to assess brain responses of RPE65 patients after stimulation of their most recently treated eye before and 1–3 years after GT. Subjects Seven RPE65 subjects who were part of the follow-on clinical trial were separately consented to participate in a longitudinal neuroimaging fMRI study. Methods All subjects underwent fMRI utilizing a 3-Tesla MRI system and a 32-channel head coil. Subjects’ cortical activations were assessed using a block design paradigm of contrast reversing checkerboard stimuli delivered using an MRI compatible video system. Main Outcome Measures The primary parameters being measured in this study are the qualitative and quantitative fMRI cortical activations produced by our subject population in response to the visual task checkerboard stimulus. Results fMRI results showed minimal or no cortical responses before GT. Significant increase in cortical activation lasting at least three years after GT was observed for all subjects. Repeated measures analysis showed significant associations between cortical activations and clinical measures such as full field light sensitivity threshold (FST) for white, red, and blue colors, visual field (VF), and pupillary light reflex (PLR). Conclusions RPE65-subjects showed intact visual pathways, which became responsive and strengthened after treatment. fMRI results independently revealed the efficacy and durability of a one-time subretinal injection. The fMRI results paralleled those recently reported during the long-term clinical evaluations of the same subjects.1 Results from this study demonstrate that fMRI may play an important role in providing complementary information to patients’ ophthalmic clinical evaluation and has utility as an outcome measure for future retinal intervention studies.

Objective: The purpose of this study was to characterize intraoperative palatal lengthening with the modified Furlow cleft palatal repair and to determine whether lengthening correlated with preoperative cleft width, cleft type, or operating surgeon. Design: Retrospective study. Setting: Academic tertiary care pediatric hospital. Patients: One hundred eighty pediatric patients undergoing primary or secondary palatoplasty using the Furlow technique. Interventions: Cleft and palatal lengths and widths were measured pre- and post-Furlow cleft palatal repair. Main Outcome Measures: Immediate postoperative percentage change in surface palate length, straight palate length, and soft palate length. Results: The average cleft widest width and width at the hard–soft palate junction were 10.2 and 9.5 mm, respectively, and varied with Veau cleft type. Following Furlow palatoplasty, lengths of the curved, straight, and soft palate increased by 7.5%, 15.8%, and 30.6%, respectively. Degree of palatal lengthening varied among surgeons and Veau cleft type but was not related to cleft width. Seven (4.0%) patients developed postoperative oronasal fistulas. Patients with a Veau IV cleft and larger cleft widths were at an increased risk for fistula formation. Conclusions: This study demonstrates that overall palatal lengthening occurs with the modified Furlow technique. Long-term follow-up studies are needed to determine the clinical relevance of these findings.

Therapeutic monoclonal antibodies (mAb) are approved for treatment of several diseases including primary immunodeficiencies, cancer, asthma, and graft transplantation. Yet, only 2 mAbs are approved for administration against infectious diseases: palivizumab (respiratory syncytial virus) and raxibacumab (inhalational anthrax). Numerous protective mAbs targeting recurring and emerging bacterial and viral pathogens have been isolated, however, the high dosage (mg/kg) and associated cost of mAb manufacturing are significant hurdles for routine therapeutic delivery. Recently we have described the development of DNA vector-encoded monoclonal antibodies (DMAbs), as a possible alternative technology. This delivery targets skeletal muscle for invivo transfection to transiently produce and secrete mAb. By optimizing gene design protective levels of antibody are produced in vivo by this technology. We show that expression can last a period of weeks. These designed DMAb formulations encoding the mAb heavy and light chain genes are delivered in vivo by intramuscular injection followed by electroporation (IM-EP). Several DMAbs were developed targeting antimicrobial resistant bacteria, a serious global health concern. Additionally, we also designed DMAbs against a range of viral infections including frequent, emerging, and neglected tropical diseases. Through a series of sequence and formulation optimizations, we are able to achieve serum levels >5ug/ mL and as high as 100ug/mL, depending on the DMAb. DMAb serum levels match the protective trough level range of their purified mAb counterparts and perform on par in functional assays (e.g. killing assays, neutralization). DMAb candidates are protective against bacterial and viral challenges in mice, illustrating functionality in vivo. Data from the challenge studies will be presented. Ongoing studies are investigating DMAb gene delivery in larger animal models including rabbits, guinea pigs, and non-human primates. DNA-delivered mAbs is a flexible platform that transforms mAb delivery, allowing for repeat administration, significantly lower production costs, and expands the utility of DNA vector technology for therapeutic gene therapy. This approach may have benefit for routine DMAb gene delivery to prevent nosocomial and community-acquired infections and can be rapidly deployed during an infectious disease outbreak.