Gene Editing for CEP290-Associated Retinal Degeneration.

BACKGROUND: CEP290-associated inherited retinal degeneration causes severe early-onset vision loss due to pathogenic variants in CEP290. EDIT-101 is a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing complex designed to treat inherited retinal degeneration caused by a specific damaging variant in intron 26 of CEP290 (IVS26 variant). METHODS: We performed a phase 1-2, open-label, single-ascending-dose study in which persons 3 years of age or older with CEP290-associated inherited retinal degeneration caused by a homozygous or compound heterozygous IVS26 variant received a subretinal injection of EDIT-101 in the worse (study) eye. The primary outcome was safety, which included adverse events and dose-limiting toxic effects. Key secondary efficacy outcomes were the change from baseline in the best corrected visual acuity, the retinal sensitivity detected with the use of full-field stimulus testing (FST), the score on the Ora-Visual Navigation Challenge mobility test, and the vision-related quality-of-life score on the National Eye Institute Visual Function Questionnaire-25 (in adults) or the Children's Visual Function Questionnaire (in children). RESULTS: EDIT-101 was injected in 12 adults 17 to 63 years of age (median, 37 years) at a low dose (in 2 participants), an intermediate dose (in 5), or a high dose (in 5) and in 2 children 9 and 14 years of age at the intermediate dose. At baseline, the median best corrected visual acuity in the study eye was 2.4 log10 of the minimum angle of resolution (range, 3.9 to 0.6). No serious adverse events related to the treatment or procedure and no dose-limiting toxic effects were recorded. Six participants had a meaningful improvement from baseline in cone-mediated vision as assessed with the use of FST, of whom 5 had improvement in at least one other key secondary outcome. Nine participants (64%) had a meaningful improvement from baseline in the best corrected visual acuity, the sensitivity to red light as measured with FST, or the score on the mobility test. Six participants had a meaningful improvement from baseline in the vision-related quality-of-life score. CONCLUSIONS: The safety profile and improvements in photoreceptor function after EDIT-101 treatment in this small phase 1-2 study support further research of in vivo CRISPR-Cas9 gene editing to treat inherited retinal degenerations due to the IVS26 variant of CEP290 and other genetic causes. (Funded by Editas Medicine and others; BRILLIANCE ClinicalTrials.gov number, NCT03872479.).

A highly efficient transgene knock-in technology in clinically relevant cell types.

Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.

Coordinated conformational changes in the V1 complex during V-ATPase reversible dissociation.

Vacuolar-type ATPases (V-ATPases) are rotary enzymes that acidify intracellular compartments in eukaryotic cells. These multi-subunit complexes consist of a cytoplasmic V1 region that hydrolyzes ATP and a membrane-embedded VO region that transports protons. V-ATPase activity is regulated by reversible dissociation of the two regions, with the isolated V1 and VO complexes becoming autoinhibited on disassembly and subunit C subsequently detaching from V1. In yeast, assembly of the V1 and VO regions is mediated by the regulator of the ATPase of vacuoles and endosomes (RAVE) complex through an unknown mechanism. We used cryogenic-electron microscopy of yeast V-ATPase to determine structures of the intact enzyme, the dissociated but complete V1 complex and the V1 complex lacking subunit C. On separation, V1 undergoes a dramatic conformational rearrangement, with its rotational state becoming incompatible for reassembly with VO. Loss of subunit C allows V1 to match the rotational state of VO, suggesting how RAVE could reassemble V1 and VO by recruiting subunit C.

A Preliminary Study of Anatomical Changes Following the Use of a Pedicled Buccal Fat Pad Flap During Primary Palatoplasty.

OBJECTIVE: The purpose of this study was to examine the surgical impact of the pedicled buccal fat pad (BFP) flap on the levator veli palatini (LVP) muscle and surrounding velopharyngeal (VP) anatomy following primary palatoplasty using magnetic resonance imaging (MRI). DESIGN: Observational, prospective. SETTING: MRI studies were completed at 3 different facilities. All participants with BFP flap were operated on by the same surgeon. PARTICIPANTS: Five pediatric participants with cleft palate with or without cleft lip (CP±L) who underwent primary palatoplasty with BFP flap placement. Comparison groups consisted of 10 participants: 5 with CP±L who did not receive the BFP flap and 5 healthy controls. INTERVENTIONS: All participants underwent nonsedated MRI 2 to 5 years postoperatively. MAIN OUTCOMES AND MEASURES: Anatomical measures of the velopharynx and LVP among the 3 participant groups. RESULTS: Median values were significantly different among groups for velar length (P = .042), effective velar length (P = .048), effective VP ratio (P = .046), LVP length (P = .021), extravelar LVP length (P = .009), and LVP origin-origin distance (P = .030). Post hoc analysis revealed a statistically significant difference between the BFP and traditional repair groups for effective VP ratio (P = .040), extravelar LVP length (P = .033), and LVP length (P = .022). CONCLUSIONS: This study provides preliminary support that the BFP flap creates a longer velum, with increased distance between the posterior hard palate and the LVP, and a larger effective VP ratio compared to traditional surgical techniques. Future research is needed to determine whether this procedure provides a more favorable mechanism for VP closure.

What is the Fate of the Pedicled Buccal Fat Pad Flap When Used During Primary Palatoplasty?

ABSTRACT: Pedicled buccal fat pad flaps have more recently been applied to primary cleft palate reconstruction, and yet the integrity of the flap and the long-term impact on the palate has not yet been studied. This case study uses magnetic resonance imaging to evaluate the composition of the soft palate 5 years after the interpositional placement of bilateral pedicled buccal fat pad flaps during primary palatoplasty. Anatomical measures are used to quantify the flap and surrounding velopharynx using magnetic resonance imaging and three-dimensional computer technology, indicating that this surgical technique may have a lasting impact for children with cleft palate.

RAVE and Rabconnectin-3 Complexes as Signal Dependent Regulators of Organelle Acidification.

The yeast RAVE (Regulator of H+-ATPase of Vacuolar and Endosomal membranes) complex and Rabconnectin-3 complexes of higher eukaryotes regulate acidification of organelles such as lysosomes and endosomes by catalyzing V-ATPase assembly. V-ATPases are highly conserved proton pumps consisting of a peripheral V1 subcomplex that contains the sites of ATP hydrolysis, attached to an integral membrane V o subcomplex that forms the transmembrane proton pore. Reversible disassembly of the V-ATPase is a conserved regulatory mechanism that occurs in response to multiple signals, serving to tune ATPase activity and compartment acidification to changing extracellular conditions. Signals such as glucose deprivation can induce release of V1 from Vo, which inhibits both ATPase activity and proton transport. Reassembly of V1 with Vo restores ATP-driven proton transport, but requires assistance of the RAVE or Rabconnectin-3 complexes. Glucose deprivation triggers V-ATPase disassembly in yeast and is accompanied by binding of RAVE to V1 subcomplexes. Upon glucose readdition, RAVE catalyzes both recruitment of V1 to the vacuolar membrane and its reassembly with Vo. The RAVE complex can be recruited to the vacuolar membrane by glucose in the absence of V1 subunits, indicating that the interaction between RAVE and the Vo membrane domain is glucose-sensitive. Yeast RAVE complexes also distinguish between organelle-specific isoforms of the Vo a-subunit and thus regulate distinct V-ATPase subpopulations. Rabconnectin-3 complexes in higher eukaryotes appear to be functionally equivalent to yeast RAVE. Originally isolated as a two-subunit complex from rat brain, the Rabconnectin-3 complex has regions of homology with yeast RAVE and was shown to interact with V-ATPase subunits and promote endosomal acidification. Current understanding of the structure and function of RAVE and Rabconnectin-3 complexes, their interactions with the V-ATPase, their role in signal-dependent modulation of organelle acidification, and their impact on downstream pathways will be discussed.

Defining steps in RAVE-catalyzed V-ATPase assembly using purified RAVE and V-ATPase subcomplexes.

The vacuolar H+-ATPase (V-ATPase) is a highly conserved proton pump responsible for the acidification of intracellular organelles in virtually all eukaryotic cells. V-ATPases are regulated by the rapid and reversible disassembly of the peripheral V1 domain from the integral membrane Vo domain, accompanied by release of the V1 C subunit from both domains. Efficient reassembly of V-ATPases requires the Regulator of the H+-ATPase of Vacuoles and Endosomes (RAVE) complex in yeast. Although a number of pairwise interactions between RAVE and V-ATPase subunits have been mapped, the low endogenous levels of the RAVE complex and lethality of constitutive RAV1 overexpression have hindered biochemical characterization of the intact RAVE complex. We describe a novel inducible overexpression system that allows purification of native RAVE and RAVE-V1 complexes. Both purified RAVE and RAVE-V1 contain substoichiometric levels of subunit C. RAVE-V1 binds tightly to expressed subunit C in vitro, but binding of subunit C to RAVE alone is weak. Neither RAVE nor RAVE-V1 interacts with the N-terminal domain of Vo subunit Vph1 in vitro. RAVE-V1 complexes, like isolated V1, have no MgATPase activity, suggesting that RAVE cannot reverse V1 inhibition generated by rotation of subunit H and entrapment of MgADP that occur upon disassembly. However, purified RAVE can accelerate reassembly of V1 carrying a mutant subunit H incapable of inhibition with Vo complexes reconstituted into lipid nanodiscs, consistent with its catalytic activity in vivo. These results provide new insights into the possible order of events in V-ATPase reassembly and the roles of the RAVE complex in each event.

Asymmetry and Positioning of the Levator Veli Palatini Muscle in Children With Repaired Cleft Palate.

Purpose The purpose of this study is to examine the differences in velopharyngeal dimensions as well as levator veli palatini (levator) muscle morphology, positioning, and symmetry of children with repaired cleft palate with velopharyngeal insufficiency (VPI), children with repaired cleft palate with complete velopharyngeal closure, and children with noncleft anatomy. Method Fifteen children ranging in age from 4 to 8 years were recruited for this study. Ten of the participants had a history of repaired cleft palate, half with documented VPI and the other half with velopharyngeal closure. Five participants with noncleft anatomy were matched for age from a normative database. The magnetic resonance imaging protocol, processing methods, and analysis are consistent with that used in previous literature. Results Regarding velopharyngeal dimensions, median values were statistically significantly different between groups for sagittal angle (p = .031) and effective velopharyngeal ratio (p = .013). With respect to the levator muscle, median values were statistically significant for average extravelar length (p = .018), thickness at midline (p = .021), and thickness between the left and right muscle bundles at the point of insertion into the velum (p = .037). Remaining measures were not statistically significant. Conclusions The levator muscle is significantly different among these three groups with respect to thickness at midline, extravelar length, and symmetry at the point of insertion into the velum. Sagittal angle and effective velopharyngeal ratio are also significantly different. Participants with repaired cleft palate and VPI displayed the greatest degree of asymmetry. Future research should control for surgical procedure type to determine the impact of surgery on the levator muscle and surrounding velopharyngeal anatomy.

Interaction between the yeast RAVE complex and Vph1-containing Vo sectors is a central glucose-sensitive interaction required for V-ATPase reassembly.

The yeast vacuolar H+-ATPase (V-ATPase) of budding yeast (Saccharomyces cerevisiae) is regulated by reversible disassembly. Disassembly inhibits V-ATPase activity under low-glucose conditions by releasing peripheral V1 subcomplexes from membrane-bound Vo subcomplexes. V-ATPase reassembly and reactivation requires intervention of the conserved regulator of H+-ATPase of vacuoles and endosomes (RAVE) complex, which binds to cytosolic V1 subcomplexes and assists reassembly with integral membrane Vo complexes. Consistent with its role, the RAVE complex itself is reversibly recruited to the vacuolar membrane by glucose, but the requirements for its recruitment are not understood. We demonstrate here that RAVE recruitment to the membrane does not require an interaction with V1 Glucose-dependent RAVE localization to the vacuolar membrane required only intact Vo complexes containing the Vph1 subunit, suggesting that the RAVE-Vo interaction is glucose-dependent. We identified a short conserved sequence in the center of the RAVE subunit Rav1 that is essential for the interaction with Vph1 in vivo and in vitro Mutations in this region resulted in the temperature- and pH-dependent growth phenotype characteristic of ravΔ mutants. However, this region did not account for glucose sensitivity of the Rav1-Vph1 interaction. We quantitated glucose-dependent localization of a GFP-tagged RAVE subunit to the vacuolar membrane in several mutants previously implicated in altering V-ATPase assembly state or glucose-induced assembly. RAVE localization did not correlate with V-ATPase assembly levels reported previously in these mutants, highlighting both the catalytic nature of RAVE's role in V-ATPase assembly and the likelihood of glucose signaling to RAVE independently of V1.

Variations in Velopharyngeal Structure in Adults With Repaired Cleft Palate.

OBJECTIVE: The purpose of this study was to examine differences in velopharyngeal structures between adults with repaired cleft palate and normal resonance and adults without cleft palate. DESIGN: Thirty-six English-speaking adults, including 6 adults (2 males and 4 females) with repaired cleft palate (M = 32.5 years of age, SD = 17.4 years) and 30 adults (15 males and 15 females) without cleft palate (M = 23.3 years of age, SD = 4.1 years), participated in the study. Fourteen velopharyngeal measures were obtained on magnetic resonance images and compared between groups (cleft and noncleft). RESULTS: After adjusting for body size and sex effects, there was a statistically significant difference between groups for 10 out of the 14 velopharyngeal measures. Compared to those without cleft palate, participants with repaired cleft palate had a significantly shorter hard palate height and length, shorter levator muscle length, shorter intravelar segment, more acute levator angles of origin, shorter and thinner velum, and greater pharyngeal depth. CONCLUSION: Although significant differences were evident in the cleft palate group, individuals displayed normal resonance. These findings suggest that a wide variability in velopharyngeal anatomy can occur in the presence of normal resonance, particularly for those with repaired cleft palate. Future research is needed to understand how anatomic variability impacts function, such as during speech.

Current Controversies in Metopic Suture Craniosynostosis.

Metopic craniosynostosis is being reported with an increasing incidence and is now the second most common type of isolated suture craniosynostosis. Numerous areas of controversy exist in the work-up and management, including defining the diagnosis in the less severe phenotype, the association with neurodevelopmental delay, the impact of surgical treatment, and the applicability of various techniques and their timing on outcomes.

Examining a New Method to Studying Velopharyngeal Structures in a Child With 22q11.2 Deletion Syndrome.

Purpose: To date, no studies have imaged the velopharynx in children with 22q11.2 deletion syndrome (22q11.2 DS) without the use of sedation. Dysmorphology in velopharyngeal structures has been shown to have significant negative implications on speech among these individuals. This single case study was designed to assess the feasibility of a child-friendly magnetic resonance imaging (MRI) scanning protocol in this clinically challenging population and to determine the utility of this MRI protocol for future work in this area. Method: One 6-year-old White girl diagnosed with 22q11.2 DS was imaged using a child-friendly, nonsedated MRI protocol. Quantitative and qualitative measures of the velopharyngeal area and associated structures were evaluated, and comparisons were made to age-matched control subjects with normal velopharyngeal anatomy. Results: MRI data were successfully obtained using the child-friendly scanning protocol in the subject in the present study. Quantitative and qualitative differences of the levator muscle and associated velopharyngeal structures were noted. Using these MRI and structural analyses methods, insights related to muscle morphology can be obtained and considered as part of the research and clinical examination of children with 22q11.2 DS. Conclusion: The imaging protocol described in this study presents an effective means to counteract difficulties in imaging young children.

Management of midface maxillofacial trauma.

The management of midface trauma continues to challenge maxillofacial surgeons. The complex local anatomy and functional and cosmetic importance of the region make precise surgical correction and reconstruction essential to success.

The effect of preoperative recombinant erythropoietin on postoperative hematocrit level after orthognathic surgery.

PURPOSE: To compare the postoperative red cell mass as indicated by the hematocrit value of orthognathic surgery patients given iron supplementation and a single preoperative dose of erythropoietin alpha (EPO) and patients who did not receive either EPO or iron supplementation. PATIENTS AND METHODS: Subjects who had a Le Fort I osteotomy (LFI) or a combination of LFI and bilateral sagittal split osteotomy between 2005 and 2008 and were aged at least 13 years were included. Subjects were excluded if they had a history of maxillofacial trauma, a craniofacial syndrome, or a major systemic medical condition. Subjects either had EPO administered with iron supplements before surgery (surgeon A protocol) or received neither (surgeon B protocol). Venous blood samples were taken, in accordance with clinic protocol, before surgery (before administration of EPO) and on postoperative day 1. Multiple linear regression with backward selection was used to analyze the change in hematocrit value. Explanatory variables included group, preoperative hematocrit level, age, gender, length of surgery, blood loss, and crystalloid (fluid replacement) volume. RESULTS: The study included 178 eligible patients: 86 (48%) had a combination of LFI and bilateral sagittal split osteotomy and 92 (52%) had an isolated LFI. Of the patients, 114 (64%) had EPO/iron supplements administered before surgery whereas 64 did not. The mean change in hematocrit level as an indicator of the change in red cell mass was statistically significantly different (P = .01) for the subjects who received preoperative administration of EPO with iron supplementation compared with those who did not receive EPO plus iron. The administration of EPO plus iron was protective: the decrease in hematocrit level after surgery was smaller for subjects in the EPO group even after we controlled for age, gender, preoperative hematocrit level, length of surgery, blood loss, and crystalloid (fluid replacement) volume. CONCLUSIONS: A single preoperative dose of erythropoietin with iron supplementation resulted in a smaller decrease, on average, in postoperative red cell mass as indicated by hematocrit value in patients with complicated orthognathic surgery procedures.

Pediatric head injuries.

Head injuries in children are common, comprising more than half of all injuries sustained. The mortality and morbidity associated with traumatic head injury in children is staggering, and the cumulative effect of such on the pediatric and general populations is propagated through related health care measures and subsequent socioeconomic burden. The majority of deaths due to trauma in children are caused by brain injury. This article reviews the evaluation and management of scalp injuries in the pediatric patient. The second portion addresses skull fractures, the specter of child abuse, management of acute fracture, and the phenomenon of growing skull fractures.

Craniofacial and orbital dermoids in children.

Dermoid cysts are congenital lesions that commonly arise from nondisjunction of surface ectoderm from deeper neuroectodermal structures. They tend to be found along planes of embryonic closure. Classification by site is helpful for diagnostic planning and surgical treatment. A distinction can be made between frontotemporal, orbital, frontoethmoidal, and calvarial lesions. The risk of extension into deeper tissues must be determined before surgical intervention. Simple lesions are amenable to direct excision. Deeper lesions often require a coordinated surgical approach between a neurosurgeon and craniofacial surgeon after thorough radiographic imaging. Follow-up through the developmental years is recommended for complex dermoid lesions.

Three-dimensional surgical simulation.

In this article, we discuss the development of methods for computer-aided jaw surgery, which allows us to incorporate the high level of precision necessary for transferring virtual plans into the operating room. We also present a complete computer-aided surgery system developed in close collaboration with surgeons. Surgery planning and simulation include construction of 3-dimensional surface models from cone-beam computed tomography, dynamic cephalometry, semiautomatic mirroring, interactive cutting of bone, and bony segment repositioning. A virtual setup can be used to manufacture positioning splints for intraoperative guidance. The system provides further intraoperative assistance with a computer display showing jaw positions and 3-dimensional positioning guides updated in real time during the surgical procedure. The computer-aided surgery system aids in dealing with complex cases with benefits for the patient, with surgical practice, and for orthodontic finishing. Advanced software tools for diagnosis and treatment planning allow preparation of detailed operative plans, osteotomy repositioning, bone reconstructions, surgical resident training, and assessing the difficulties of the surgical procedures before the surgery. Computer-aided surgery can make the elaboration of the surgical plan a more flexible process, increase the level of detail and accuracy of the plan, yield higher operative precision and control, and enhance documentation of cases.