404 results on '"Wolfe Sa"'
Search Results
52. THE VALUE OF SILVER AS A DRESSING
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Wolfe Sa
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Silver ,business.industry ,Statistics ,Humans ,Medicine ,Surgery ,business ,Bandages ,Value (mathematics) - Published
- 1986
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53. The Orbit—Cone or Pyramid?
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Wolfe Sa
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Adult ,Cone (topology) ,business.industry ,Pyramid ,Humans ,Medicine ,Surgery ,Geometry ,Orbit (control theory) ,business ,Orbit - Published
- 1980
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54. Charge of the Bite Brigade
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Wolfe Sa
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Cleft Palate ,Insurance Claim Reporting ,Insurance ,Fees, Medical ,business.industry ,Electrical engineering ,Humans ,Medicine ,Surgery ,Charge (physics) ,Surgery, Plastic ,business - Published
- 1988
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55. Study protocol: a dissemination trial of computerized psychological treatment for depression and alcohol/other drug use comorbidity in an Australian clinical service
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Kay-Lambkin Frances J, Baker Amanda L, Healey Alison, Wolfe Samantha, Simpson Aaron, Brooks Michelle, Bowman Jenny, and Childs Steven
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Psychiatry ,RC435-571 - Abstract
Abstract Background The rise of the internet and related technologies has significant implications for the treatment of complex health problems, including the combination of depression and alcohol/other drug (AOD) misuse. To date, no research exists to test the real world uptake of internet and computer-delivered treatment programs in clinical practice. This study is important, as it is the first to examine the adoption of the SHADE treatment program, a DVD-based psychological treatment for depression and AOD use comorbidity, by clinicians working in a publicly-funded AOD clinical service. The study protocol that follows describes the methodology of this dissemination trial. Methods/design 19 clinicians within an AOD service on the Central Coast of New South Wales, Australia, will be recruited to the trial. Consenting clinicians will participate in a baseline focus group discussion designed to explore their experiences and perceived barriers to adopting innovation in their clinical practice. Computer comfort and openness to innovation will also be assessed. Throughout the trial, current, new and wait-list clients will be referred to the research program via the clinical service, which will involve clients completing a baseline and 15-week follow-up clinical assessment with independent research assistants, comprising a range of mental health and AOD measures. Clinicians will also complete session checklists following each clinical session with a client, outlining the extent to which the SHADE computer program was used. Therapeutic alliance will be measured at intake and discharge from both the clinician and client perspectives. Discussion This study will provide comprehensive data on the factors associated with the adoption of an innovative, computer-delivered evidence-based treatment program, SHADE, by clinicians working in an AOD service. The results will contribute to the development of a model of dissemination of SHADE, which could be applied to a range of technological innovations. Clinical trials registry Australian Clinical Trial Registration Number: ACTRN12611000382976.
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- 2012
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56. BCL11A +58/+55 enhancer-editing facilitates HSPC engraftment and HbF induction in rhesus macaques conditioned with a CD45 antibody-drug conjugate.
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Demirci S, Zeng J, Palchaudhuri R, Wu C, Abraham DM, Hayal TB, Essawi K, Nguyen MA, Stasula U, Chu R, Leonard A, Porter SN, Khan MBN, Hinojosa G, Uchida N, Hong S, Lazzarotto CR, Neri NR, da Silva LF, Pellin D, Verma A, Lanieri L, Bhat A, Hammond K, Tate T, Maitland SA, Sheikhsaran F, Bonifacino AC, Krouse AE, Linde NS, Engels T, Golomb J, Tsai SQ, Pruett-Miller SM, Scadden DT, Dunbar CE, Wolfe SA, Donahue RE, Olson LM, Bauer DE, and Tisdale JF
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Editing the +58 region of the BCL11A erythroid enhancer has shown promise in treating β-globin disorders. To address variations in fetal hemoglobin (HbF) response, we investigated editing both +58 and +55 enhancers. Rhesus macaques transplanted with edited hematopoietic stem/progenitor cells (HSPCs) following busulfan conditioning exhibited durable, high-level (∼90%) editing frequencies post transplantation with sustained HbF reactivation over 4 years, without hematological perturbations. HbF levels were further boosted by stress erythropoiesis or hydroxyurea. Bone marrow analysis revealed that gene edits were predominantly programmed deletions, programmed inversions, and short indels, each disrupting the enhancer core TGN
7-9 WGATAR half E-box/GATA binding motifs. Nonprogrammed long deletions were disfavored in engrafting cells. CD45 antibody-drug conjugate (ADC) conditioning achieved comparable engraftment and HbF reactivation, whereas lentiviral vector tracking showed polyclonal reconstitution with dynamics similar to animals conditioned with total body irradiation (TBI) or busulfan. Joining CD45-ADC conditioning with combined enhancer editing presents an effective strategy for β-hemoglobinopathies, enabling durable HbF reactivation without chemotherapy., Competing Interests: Declaration of interests R.P., L.L., A.B., K.H., T.T., and L.M.O. were employees of Magenta Therapeutics at the time of the initiation of the project., (Published by Elsevier Inc.)- Published
- 2024
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57. The co-receptor Neuropilin-1 enhances proliferation in inv(16) acute myeloid leukemia via VEGF signaling.
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Hegde M, Ahmad MH, Mulet Lazaro R, Sugita M, Li R, Hu K, Gebhard C, Guzman ML, Bushweller JH, Zhu LJ, Brehm M, Wolfe SA, Delwel R, and Castilla LH
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Oncogenic programs regulate the proliferation and maintenance of cancer stem cells, and can define pharmacologic dependencies. In acute myeloid leukemia (AML) with the chromosome inversion 16 (inv(16)), the fusion oncoprotein CBFβ::MYH11 regulates pathways associated with leukemia stem cell activity. Here we demonstrate that expression of Neuropilin-1 (NRP1) is regulated by the fusion oncoprotein, and promotes AML expansion. Mechanistically, we show that the NRP1 locus has open chromatin in inv(16) AML, and that CBFβ::MYH11 modulates the local function of the transcription factors ERG, GATA2 and RUNX1 to sustain NRP1 levels. We found that ERG activates NRP1 expression, and that CBFβ::MYH11 knockdown represses ERG expression, thereby allowing the repressive activity of GATA2/RUNX1 at three NRP1 enhancers. Functionally, we demonstrate that NRP1 enhances the expansion of leukemic cells in vitro and in mice, and that this activity is dependent on its VEGFR-associated FV/FVIII domain. Finally, we show that treatment with VEGF inhibitor axitinib reduces AML cell growth and delays median leukemia latency in vivo. Our findings reveal that the NRP1/VEGF axis mediates proliferation in inv(16) AML blasts, and suggest that targeting NRP1 function could be promising in combination AML therapy., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)
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- 2024
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58. A brown fat-enriched adipokine, ASRA, is a leptin receptor antagonist that stimulates appetite.
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Huang L, Liu P, Du Y, Bazan JF, Pan D, Chen Q, Lee A, Kola VSR, Wolfe SA, and Wang YX
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The endocrine control of food intake remains incompletely understood, and whether the leptin receptor (LepR)-mediated anorexigenic pathway in the hypothalamus is negatively regulated by a humoral factor is unknown. Here, we identify an appetite-stimulating factor - ASRA - that represents a peripheral signal of energy deficit and orthosterically antagonizes LepR signaling. Asra encodes an 8 kD protein that is abundantly and selectively expressed in adipose tissue and to a lesser extent, in liver. ASRA associates with autophagy vesicles and its secretion is enhanced by energy deficiency. In vivo, fasting and cold stimulate Asra expression and increase its protein concentration in cerebrospinal fluid. Asra overexpression attenuates LepR signaling, leading to elevated blood glucose and development of severe hyperphagic obesity. Conversely, either adipose- or liver-specific Asra knockout mice display increased leptin sensitivity, improved glucose homeostasis, reduced food intake, resistance to high-fat diet-induced obesity, and blunted cold-evoked feeding response. Mechanistically, ASRA acts as a high affinity antagonist of LepR. AlphaFold2-multimer prediction and mutational studies suggest that a core segment of ASRA binds to the immunoglobin-like domain of LepR, similar to the 'site 3' recognition of the A-B loop of leptin. While administration of recombinant wild-type ASRA protein promotes food intake and increases blood glucose in a LepR signaling-dependent manner, point mutation within ASRA that disrupts LepR-binding results in a loss of these effects. Our studies reveal a previously unknown endocrine mechanism in appetite regulation and have important implications for our understanding of leptin resistance., Competing Interests: Competing interests Y.-X.W, L.H., and Y.D. have filed a patent application on ASRA.
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- 2024
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59. Increasing intracellular dNTP levels improves prime editing efficiency.
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Liu P, Ponnienselvan K, Nyalile T, Oikemus S, Joynt AT, Iyer S, Kelly K, Guo D, Kyawe PP, Vanderleeden E, Redick SD, Huang L, Chen Z, Lee JM, Schiffer CA, Harlan DM, Wang JP, Emerson CP Jr, Lawson ND, Watts JK, Sontheimer EJ, Luban J, and Wolfe SA
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In primary cell types, intracellular deoxynucleotide triphosphate (dNTP) levels are tightly regulated in a cell cycle-dependent manner. We report that prime editing efficiency is increased by mutations that improve the enzymatic properties of Moloney murine leukemia virus reverse transcriptase and treatments that increase intracellular dNTP levels. In combination, these modifications produce substantial increases in precise editing rates., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)
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- 2024
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60. A Simplified Approach for Surgical Correction of Vertical Orbital Dystopia: A 45-Year Retrospective Cohort Study.
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Wolfe EM, Ainuz BY, Ragheb J, and Wolfe SA
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- Humans, Retrospective Studies, Female, Male, Middle Aged, Adult, Craniotomy methods, Craniotomy adverse effects, Treatment Outcome, Postoperative Complications epidemiology, Postoperative Complications etiology, Orbital Diseases surgery, Orbital Diseases etiology, Osteotomy methods, Osteotomy adverse effects, Plastic Surgery Procedures methods, Plastic Surgery Procedures adverse effects, Follow-Up Studies, Facial Asymmetry surgery, Facial Asymmetry etiology, Orbit surgery
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Background: Vertical orbital dystopia (VOD) results in significant facial asymmetry, psychological distress, and poor quality of life in affected patients. The traditional approach (TA) for surgical correction has entailed a standard frontal craniotomy along with circumferential orbital osteotomy, vertical translocation of the orbit, and bone grafting to the lower maxilla. Caution has been expressed regarding its invasive transcranial nature. In this report, the authors describe the limited approach (LA) for simplified surgical correction of VOD, which obviates the need for a standard frontal craniotomy., Methods: A 45-year retrospective review was conducted of all patients who underwent surgical correction of VOD, as performed by a single surgeon. Demographic details, procedural characteristics, and complications were compared between patients who underwent correction by the TA and those who underwent correction by the LA. Complications were defined as cerebrospinal fluid leak, infection of the frontal bone, permanent diplopia, permanent ptosis, sudden-onset vision loss, persistent asymmetry, and surgical revision., Results: Forty patients met inclusion criteria for correction of true VOD, of which 18 underwent the TA and 22 underwent the LA. Mean length of hospital stay was 5.3 ± 2.3 days and 4.0 ± 1.5 days for the TA and LA cohorts, respectively. Mean follow-up time was 4.9 ± 7.5 years for the TA cohort and 2.6 ± 3.3 years for the LA cohort. The only reported complications were persistent asymmetry in 2 patients in the TA cohort, with 1 patient requiring surgical revision because of undercorrection, whereas the LA cohort exhibited no postoperative asymmetry or need for surgical revision., Conclusions: Both the TA and the LA are effective for surgical correction of VOD. The limited craniotomy of the LA reduces exposure of intracranial structures and adequately achieves postoperative symmetry., Clinical Question/level of Evidence: Therapeutic, III., (Copyright © 2023 by the American Society of Plastic Surgeons.)
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- 2024
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61. PRC1.6 localizes on chromatin with the human silencing hub (HUSH) complex for promoter-specific silencing.
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Rodríguez TC, Yurkovetskiy L, Nagalekshmi K, Lam CHO, Jazbec E, Maitland SA, Wolfe SA, Sontheimer EJ, and Luban J
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An obligate step in the life cycle of HIV-1 and other retroviruses is the establishment of the provirus in target cell chromosomes. Transcriptional regulation of proviruses is complex, and understanding the mechanisms underlying this regulation has ramifications for fundamental biology, human health, and gene therapy implementation. The three core components of the Human Silencing Hub (HUSH) complex, TASOR, MPHOSPH8 (MPP8), and PPHLN1 (Periphilin 1), were identified in forward genetic screens for host genes that repress provirus expression. Subsequent loss-of-function screens revealed accessory proteins that collaborate with the HUSH complex to silence proviruses in particular contexts. To identify proteins associated with a HUSH complex-repressed provirus in human cells, we developed a technique, Provirus Proximal Proteomics, based on proximity labeling with C-BERST (dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging). Our screen exploited a lentiviral reporter that is silenced by the HUSH complex in a manner that is independent of the integration site in chromatin. Our data reveal that proviruses silenced by the HUSH complex are associated with DNA repair, mRNA processing, and transcriptional silencing proteins, including L3MBTL2, a member of the non-canonical polycomb repressive complex 1.6 (PRC1.6). A forward genetic screen confirmed that PRC1.6 components L3MBTL2 and MGA contribute to HUSH complex-mediated silencing. PRC1.6 was then shown to silence HUSH-sensitive proviruses in a promoter-specific manner. Genome wide profiling showed striking colocalization of the PRC1.6 and HUSH complexes on chromatin, primarily at sites of active promoters. Finally, PRC1.6 binding at a subset of genes that are silenced by the HUSH complex was dependent on the core HUSH complex component MPP8. These studies offer new tools with great potential for studying the transcriptional regulation of proviruses and reveal crosstalk between the HUSH complex and PRC1.6., Competing Interests: COMPETING INTERESTS The authors declare no competing interests.
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- 2024
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62. Long-term Quality of Life After Thyroidectomy: Transoral Endoscopic Thyroidectomy Vestibular Approach Versus Transcervical Approach.
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Nagururu NV, Seo S, Ding AS, Grogan R, Wolfe SA, Harbison RA, Tufano RP, and Russell JO
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- Humans, Male, Female, Adult, Prospective Studies, Middle Aged, Natural Orifice Endoscopic Surgery methods, Surveys and Questionnaires, Quality of Life, Thyroidectomy methods
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Objective: To compare long-term health-related quality of life (HRQOL) after Transoral Endoscopic Thyroidectomy Vestibular Approach (TOETVA) and transcervical approach (TCA) thyroidectomy., Study Design: Prospective cohort study., Setting: Tertiary referral center., Methods: A web-based survey was distributed to patients at our institution who met the criteria for TOETVA and underwent thyroidectomy by TOETVA or TCA between August 2017 and October 2021. All survey participants were at least 6 months postsurgery. Minors, non-English speakers, and patients who received concomitant neck dissection or reoperative thyroidectomy were excluded from the study. The survey assessed quality of life through 4 standardized instruments: the Dermatology Life Quality Index (DLQI), the Eating Assessment Tool (EAT-10), the Voice Handicap Index (VHI-10), and the Short Form Health Survey (SF-36)., Results: A total of 108 TOETVA and 129 TCA patients were included in the study. The median age of respondents was 44 (36, 54; 25th, 75th percentile) years and median time from surgery to survey was 35 (22, 45; 25th, 75th percentile) months. TOETVA group DLQI (0.63 vs 0.99; P = .17), VHI-10 (1.94 vs 1.67; P = .35), EAT-10 (2.14 vs 2.32; P = .29), SF-36 physical component (52.25 vs 51.00; P = .25), and SF-36 mental component (47.74 vs 47.29; P = .87) scores were all similar to those of the TCA group. Scrutinizing specific DLQI questions, individuals in the TOETVA group were less self-conscious of their skin as compared to the TCA group (Q2; 0.08 vs 0.26, P = .03)., Conclusion: Long-term HRQOL after TOETVA is similar to TCA, with significantly lower skin-related self-consciousness., (© 2024 The Authors. Otolaryngology–Head and Neck Surgery published by Wiley Periodicals LLC on behalf of American Academy of Otolaryngology–Head and Neck Surgery Foundation.)
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- 2024
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63. An Oncoplastic Approach to Primary Pediatric Pterygomaxillary Osteosarcoma.
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Ramirez AL, Townsend AN, Weber L, Piccinini PS, Wolfe EM, Taylor MW, Haglund TA, Shraiteh MA, Hannan R, Fader ME, Ragheb J, Wolfe SA, and Steinberg JP
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- Humans, Child, Male, Tomography, X-Ray Computed, Osteosarcoma surgery
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Osteosarcomas arising within the pterygomaxillary/infratemporal fossa region are rare among the pediatric population. Survival rates are most influenced by tumor resection with negative margins, which can be dependent on surgical accessibility of the tumor site. The pterygomaxillary/infratemporal fossa location poses several challenges to safe and adequate tumor resection, including proximity of the facial nerve and great vessels and scarring associated with traditional transfacial approaches. In this article, we present the case of a 6-year-old boy with an osteosarcoma of the left pterygomaxillary/infratemporal fossa region successfully managed with an "oncoplastic" approach, incorporating the use of CAD/CAM and mixed reality technologies., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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- 2024
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64. Ex vivo culture resting time impacts transplantation outcomes of genome-edited human hematopoietic stem and progenitor cells in xenograft mouse models.
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Demirci S, Khan MBN, Hinojosa G, Le A, Leonard A, Essawi K, Gudmundsdottir B, Liu X, Zeng J, Inam Z, Chu R, Uchida N, Araki D, London E, Butt H, Maitland SA, Bauer DE, Wolfe SA, Larochelle A, and Tisdale JF
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- Animals, Humans, Mice, CRISPR-Cas Systems genetics, Electroporation methods, Heterografts, Cell Survival, Antigens, CD34 metabolism, Repressor Proteins genetics, Repressor Proteins metabolism, Gene Editing methods, Hematopoietic Stem Cells metabolism, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cell Transplantation methods
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Ex vivo resting culture is a standard procedure following genome editing in hematopoietic stem and progenitor cells (HSPCs). However, prolonged culture may critically affect cell viability and stem cell function. We investigated whether varying durations of culture resting times impact the engraftment efficiency of human CD34+ HSPCs edited at the BCL11A enhancer, a key regulator in the expression of fetal hemoglobin. We employed electroporation to introduce CRISPR-Cas9 components for BCL11A enhancer editing and compared outcomes with nonelectroporated (NEP) and electroporated-only (EP) control groups. Post-electroporation, we monitored cell viability, death rates, and the frequency of enriched hematopoietic stem cell (HSC) fractions (CD34+CD90+CD45RA- cells) over a 48-hour period. Our findings reveal that while the NEP group showed an increase in cell numbers 24 hours post-electroporation, both EP and BCL11A-edited groups experienced significant cell loss. Although CD34+ cell frequency remained high in all groups for up to 48 hours post-electroporation, the frequency of the HSC-enriched fraction was significantly lower in the EP and edited groups compared to the NEP group. In NBSGW xenograft mouse models, both conditioned with busulfan and nonconditioned, we found that immediate transplantation post-electroporation led to enhanced engraftment without compromising editing efficiency. Human glycophorin A+ (GPA+) red blood cells (RBCs) sorted from bone marrow of all BCL11A edited mice exhibited similar levels of γ-globin expression, regardless of infusion time. Our findings underscore the critical importance of optimizing the culture duration between genome editing and transplantation. Minimizing this interval may significantly enhance engraftment success and minimize cell loss without compromising editing efficiency. These insights offer a pathway to improve the success rates of genome editing in HSPCs, particularly for conditions like sickle cell disease., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Published by Elsevier Inc.)
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- 2024
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65. Transoral endoscopic vestibular approach Sistrunk procedure (TEVAS)-case report and scoping review.
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Nakai MY, Tenório LR, Oliveira JDSC, Ghirardello LB, Cavalcanti IR, Russell JO, Wolfe SA, Banuchi VE, Menezes MB, and Gonçalves AJ
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Background: Thyroglossal duct cyst (TGDC) is the most common congenital neck mass among the pediatric population. Less than 10% of the cases occur in adults. The standard of care for TGDC is surgical treatment with the Sistrunk procedure via a traditional transverse cervicotomy. This technique involves the resection of the cyst with its tract and the central portion of the hyoid bone body to avoid recurrence. The transoral vestibular approach has gained popularity as an alternative approach to open neck surgery in order to eliminate the transcervical scar associated with these procedures., Methods: We describe a case of an endoscopic Sistrunk procedure performed by the transoral vestibular approach. A scoping review of the transoral endoscopic vestibular approach Sistrunk procedure (TEVAS) was performed. The PubMed, Medline, Cochrane, Lilacs, Scielo, Mary Ann Libert and Scopus databases were systematically searched by using a Medical Subject Heading (MeSH)-optimized search strategy. The selection of papers followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines after setting inclusion and exclusion criteria., Results: The case was successful and without complications. Five studies were included in the final analysis for this review., Conclusions: This novel approach to the Sistrunk procedure is an effective alternative way to treat TGDC in selected patients who are motivated to avoid a visible neck scar., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://gs.amegroups.com/article/view/10.21037/gs-23-357/coif). M.Y.N. is a speaker for DMC. J.O.R. is a consultant for Baxter scientific and does expert testimony for Baxter scientific. The other authors have no conflicts of interest to declare., (2024 Gland Surgery. All rights reserved.)
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- 2024
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66. Direct delivery of stabilized Cas-embedded base editors achieves efficient and accurate editing of clinically relevant targets.
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Lee JM, Zeng J, Liu P, Nguyen MA, Loustaunau DS, Bauer DE, Yilmaz NK, Wolfe SA, and Schiffer CA
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Over the last 5 years, cytosine base editors (CBEs) have emerged as a promising therapeutic tool for specific editing of single nucleotide variants and disrupting specific genes associated with disease. Despite this promise, the currently available CBE's have the significant liabilities of off-target and bystander editing activities, in part due to the mechanism by which they are delivered, causing limitations in their potential applications. In this study we engineeredhighly stabilized Cas-embedded CBEs (sCE_CBEs) that integrate several recent advances, andthat are highly expressible and soluble for direct delivery into cells as ribonucleoprotein (RNP) complexes. Our resulting sCE_CBE RNP complexes efficiently and specifically target TC dinucleotides with minimal off-target or bystander mutations. Additional uracil glycosylase inhibitor (UGI) protein in trans further increased C-to-T editing efficiency and target purity in a dose-dependent manner, minimizing indel formation to untreated levels. A single electroporation was sufficient to effectively edit the therapeutically relevant locus for sickle cell disease in hematopoietic stem and progenitor cells (HSPC) in a dose dependent manner without cellular toxicity. Significantly, these sCE_CBE RNPs permitted for the transplantation of edited HSPCs confirming highly efficient editing in engrafting hematopoietic stem cells in mice. The success of the designed sCBE editors, with improved solubility and enhanced on-target editing, demonstrates promising agents for cytosine base editing at other disease-related sites in HSPCs and other cell types.
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- 2024
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67. Self-delivering, chemically modified CRISPR RNAs for AAV co-delivery and genome editing in vivo.
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Zhang H, Kelly K, Lee J, Echeverria D, Cooper D, Panwala R, Amrani N, Chen Z, Gaston N, Wagh A, Newby GA, Xie J, Liu DR, Gao G, Wolfe SA, Khvorova A, Watts JK, and Sontheimer EJ
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- Animals, Mice, Tissue Distribution, RNA genetics, Oligonucleotides, Gene Editing, RNA, Guide, CRISPR-Cas Systems
- Abstract
Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a 'protecting oligo'), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)
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- 2024
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68. Runx1-R188Q germ line mutation induces inflammation and predisposition to hematologic malignancies in mice.
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Ahmad MH, Hegde M, Wong WJ, Mohammadhosseini M, Garrett L, Carrascoso A, Issac N, Ebert B, Silva JC, Pihan G, Zhu LJ, Wolfe SA, Agarwal A, Liu PP, and Castilla LH
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- Animals, Mice, Humans, Germ-Line Mutation, Core Binding Factor Alpha 2 Subunit genetics, Core Binding Factor Alpha 2 Subunit metabolism, Disease Susceptibility, Inflammation genetics, DNA, Blood Platelet Disorders genetics, Hematologic Neoplasms genetics, Hematologic Neoplasms complications
- Abstract
Germ line mutations in the RUNX1 gene cause familial platelet disorder (FPD), an inherited disease associated with lifetime risk to hematopoietic malignancies (HM). Patients with FPD frequently show clonal expansion of premalignant cells preceding HM onset. Despite the extensive studies on the role of RUNX1 in hematopoiesis, its function in the premalignant bone marrow (BM) is not well-understood. Here, we characterized the hematopoietic progenitor compartments using a mouse strain carrying an FPD-associated mutation, Runx1R188Q. Immunophenotypic analysis showed an increase in the number of hematopoietic stem and progenitor cells (HSPCs) in the Runx1R188Q/+ mice. However, the comparison of Sca-1 and CD86 markers suggested that Sca-1 expression may result from systemic inflammation. Cytokine profiling confirmed the dysregulation of interferon-response cytokines in the BM. Furthermore, the expression of CD48, another inflammation-response protein, was also increased in Runx1R188Q/+ HSPCs. The DNA-damage response activity of Runx1R188Q/+ hematopoietic progenitor cells was defective in vitro, suggesting that Runx1R188Q may promote genomic instability. The differentiation of long-term repopulating HSCs was reduced in Runx1R188Q/+ recipient mice. Furthermore, we found that Runx1R188Q/+ HSPCs outcompete their wild-type counterparts in bidirectional repopulation assays, and that the genetic makeup of recipient mice did not significantly affect the clonal dynamics under this setting. Finally, we demonstrate that Runx1R188Q predisposes to HM in cooperation with somatic mutations found in FPDHM, using 3 mouse models. These studies establish a novel murine FPDHM model and demonstrate that germ line Runx1 mutations induce a premalignant phenotype marked by BM inflammation, selective expansion capacity, defective DNA-damage response, and predisposition to HM., (Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution.)
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- 2023
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69. Generation and application of endogenously floxed alleles for cell-specific knockout in zebrafish.
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Shin M, Yin HM, Shih YH, Nozaki T, Portman D, Toles B, Kolb A, Luk K, Isogai S, Ishida K, Hanasaka T, Parsons MJ, Wolfe SA, Burns CE, Burns CG, and Lawson ND
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- Mice, Animals, Mice, Knockout, Alleles, Gene Knockout Techniques, Zebrafish genetics, Integrases genetics
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The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for routine generation and application of endogenously floxed alleles in zebrafish., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)
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- 2023
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70. Addressing the dNTP bottleneck restricting prime editing activity.
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Ponnienselvan K, Liu P, Nyalile T, Oikemus S, Joynt AT, Kelly K, Guo D, Chen Z, Lee JM, Schiffer CA, Emerson CP, Lawson ND, Watts JK, Sontheimer EJ, Luban J, and Wolfe SA
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Prime editing efficiency is modest in cells that are quiescent or slowly proliferating where intracellular dNTP levels are tightly regulated. MMLV-reverse transcriptase - the prime editor polymerase subunit - requires high intracellular dNTPs levels for efficient polymerization. We report that prime editing efficiency in primary cells and in vivo is increased by mutations that enhance the enzymatic properties of MMLV-reverse transcriptase and can be further complemented by targeting SAMHD1 for degradation.
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- 2023
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71. Crosstalk between corepressor NRIP1 and cAMP signaling on adipocyte thermogenic programming.
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Tsagkaraki E, Guilherme A, Nicoloro SM, Kelly M, Lifshitz LM, Wang H, Min K, Rowland LA, Santos KB, Wetoska N, Friedline RH, Maitland SA, Chen M, Weinstein LS, Wolfe SA, Kim JK, and Czech MP
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- Mice, Humans, Animals, Nuclear Receptor Interacting Protein 1 metabolism, Mice, Obese, Obesity metabolism, Thermogenesis genetics, Adipocytes metabolism, Signal Transduction
- Abstract
Objectives: Nuclear receptor interacting protein 1 (NRIP1) suppresses energy expenditure via repression of nuclear receptors, and its depletion markedly elevates uncoupled respiration in mouse and human adipocytes. We tested whether NRIP1 deficient adipocytes implanted into obese mice would enhance whole body metabolism. Since β-adrenergic signaling through cAMP strongly promotes adipocyte thermogenesis, we tested whether the effects of NRIP1 knock-out (NRIP1KO) require the cAMP pathway., Methods: NRIP1KO adipocytes were implanted in recipient high-fat diet (HFD) fed mice and metabolic cage studies conducted. The Nrip1 gene was disrupted by CRISPR in primary preadipocytes isolated from control vs adipose selective GsαKO (cAdGsαKO) mice prior to differentiation to adipocytes. Protein kinase A inhibitor was also used., Results: Implanting NRIP1KO adipocytes into HFD fed mice enhanced whole-body glucose tolerance by increasing insulin sensitivity, reducing adiposity, and enhancing energy expenditure in the recipients. NRIP1 depletion in both control and GsαKO adipocytes was equally effective in upregulating uncoupling protein 1 (UCP1) and adipocyte beiging, while β-adrenergic signaling by CL 316,243 was abolished in GsαKO adipocytes. Combining NRIP1KO with CL 316,243 treatment synergistically increased Ucp1 gene expression and increased the adipocyte subpopulation responsive to beiging. Estrogen-related receptor α (ERRα) was dispensable for UCP1 upregulation by NRIPKO., Conclusions: The thermogenic effect of NRIP1 depletion in adipocytes causes systemic enhancement of energy expenditure when such adipocytes are implanted into obese mice. Furthermore, NRIP1KO acts independently but cooperatively with the cAMP pathway in mediating its effect on adipocyte beiging., Competing Interests: Declaration of competing interest The authors declare the following competing Interests: M.P.C. and A.G. are inventors of granted US Patent US-8519118-B2, “RIP140 regulation of glucose transport”, and of granted US Patent US-8093223-B2, “RIP140 regulation of diabetes”, related to data in this manuscript on Nrip1/RIP140-depleted mouse and human adipocytes. M.P.C., E.T., and S.M.N. are inventors of US Patent US-20220220461-A1 and PCT WO2022076812A3, “Targeting Nrip1 to Alleviate Metabolic Disease”, related to CRISPR-based depletion of NRIP1 in mouse adipocytes in this manuscript. The University of Massachusetts is the grantee or potential grantee of all the above. M.P.C. declares that he is bound by a confidentiality agreement that prevents him from disclosing a potentially competing interest in this work. S.A.W. is a consultant for Chroma Medicine. The remaining authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)
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- 2023
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72. TOETVA parathyroid autofluorescence detection: hANDY-i endoscopy attachment.
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Seo S, Ali KM, Wolfe SA, Nagururu NV, Ding AS, Desai D, Harbison RA, Kim Y, Ning B, Cha RJ, and Russell JO
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- Humans, Parathyroid Glands diagnostic imaging, Parathyroid Glands surgery, Endoscopy, Gastrointestinal, Thyroid Gland diagnostic imaging, Thyroid Gland surgery, Thyroidectomy adverse effects, Hypocalcemia diagnosis, Hypocalcemia etiology
- Abstract
Background: Treatment options for thyroid pathologies have expanded to include scarless and remote access methods such as the transoral endoscopic thyroidectomy vestibular approach (TOETVA). Currently, no standardized methods exist for locating parathyroid glands (PGs) in patients undergoing TOETVA, which can lead to parathyroid injury and subsequent hypocalcemia. This early feasibility study describes and evaluates the hANDY-i endoscopic attachment for detecting PGs in transoral thyroidectomy., Methods: We used a prototype parathyroid autofluorescence imager (hANDY-i) that was mounted to a 10-mm 0-degree endoscope. The device delivers a split screen view of Red-green-blue (RGB) and near-infrared autofluorescence (NIRAF) which allows for simultaneous anatomical localization and fluorescence visualization of PGs during endoscopic thyroid dissection., Results: One cadaveric case and two patient cases were included in this study. The endoscopic hANDY-i imaging system successfully visualized PGs during all procedures., Conclusion: The ability to leverage parathyroid autofluorescence during TOETVA may lead to improved PG localization and preservation. Further human studies are needed to assess its effect on postoperative hypocalcemia and hypoparathyroidism., Competing Interests: YK is an employee of Optosurgical LLC. RC has ownership stake in Optosurgical LLC. JR is a consultant for Baxter Scientific. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Seo, Ali, Wolfe, Nagururu, Ding, Desai, Harbison, Kim, Ning, Cha and Russell.)
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- 2023
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73. Transcriptional and chromatin profiling of human blood innate lymphoid cell subsets sheds light on HIV-1 pathogenesis.
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Wang Y, Lifshitz L, Silverstein NJ, Mintzer E, Luk K, StLouis P, Brehm MA, Wolfe SA, Deeks SG, and Luban J
- Subjects
- Humans, Mice, Animals, Immunity, Innate, Lymphocytes metabolism, Interleukin-2 metabolism, Chromatin, Killer Cells, Natural, Cytokines, HIV-1 metabolism, HIV Infections genetics
- Abstract
Innate lymphoid cells (ILCs) are a diverse population of cells that include NK cells and contribute to tissue homeostasis and repair, inflammation, and provide protection from infection. The interplay between human blood ILCs, as well as their responses to HIV-1 infection, remains poorly understood. This study used transcriptional and chromatin profiling to explore these questions. Transcriptional profiling and flow cytometry analysis support that there are four main ILC subsets found in human blood. Unlike in mice, human NK cells expressed the tissue repair protein amphiregulin (AREG). AREG production was induced by TCF7/WNT, IL-2, and IL-15, and inhibited by TGFB1, a cytokine increased in people living with HIV-1. In HIV-1 infection, the percentage of AREG
+ NK cells correlated positively with the numbers of ILCs and CD4+ T cells but negatively with the concentration of inflammatory cytokine IL-6. NK-cell knockout of the TGFB1-stimulated WNT antagonist RUNX3 increased AREG production. Antiviral gene expression was increased in all ILC subsets from HIV-1 viremic people, and anti-inflammatory gene MYDGF was increased in an NK-cell subset from HIV-1-infected people whose viral load was undetectable in the absence of antiretroviral therapy. The percentage of defective NK cells in people living with HIV-1 correlated inversely with ILC percentage and CD4+ T-cell counts. CD4+ T cells and their production of IL-2 prevented the loss of NK-cell function by activating mTOR. These studies clarify how ILC subsets are interrelated and provide insight into how HIV-1 infection disrupts NK cells, including an uncharacterized homeostatic function in NK cells., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)- Published
- 2023
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74. Parathyroid gland detection using an intraoperative autofluorescence handheld imager - early feasibility study.
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Ali KM, Wolfe SA, Nagururu NV, Seo S, Han SM, Kim Y, Oh E, Kim DY, Ning B, Lee SY, Cha RJ, Tufano RP, and Russell JO
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- Humans, Parathyroid Glands diagnostic imaging, Parathyroid Glands surgery, Feasibility Studies, Thyroidectomy adverse effects, Thyroidectomy methods, Optical Imaging methods, Hypocalcemia, Hypoparathyroidism diagnosis
- Abstract
Introduction: Parathyroid glands may be compromised during thyroid surgery which can lead to hypoparathyroidism and hypocalcemia. Identifying the parathyroid glands relies on the surgeon's experience and the only way to confirm their presence was through tissue biopsy. Near infrared autofluorescence technology offers an opportunity for real-time, non-invasive identification of the parathyroid glands., Methods: We used a new research prototype (hANDY-I) developed by Optosurgical, LLC. It offers coaxial excitation light and a dual-Red Green Blue/Near Infrared sensor that guides anatomical landmarks and can aid in identification of parathyroid glands by showing a combined autofluorescence and colored image simultaneously., Results: We tested the imager during 23 thyroid surgery cases, where initial clinical feasibility data showed that out of 75 parathyroid glands inspected, 71 showed strong autofluorescence signal and were correctly identified (95% accuracy) by the imager., Conclusions: The hANDY-I prototype demonstrated promising results in this feasibility study by aiding in real-time visualization of the parathyroid glands. However, further testing by conducting randomized clinical trials with a bigger sample size is required to study the effect on levels of hypoparathyroidism and hypocalcemia., Competing Interests: Author SH was employed by i2KIE LLC. Author RC has an ownership interest in Optosurgical, LLC. Authors EO and YK were employees of Optosurgical, LLC when the study was carried out. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ali, Wolfe, Nagururu, Seo, Han, Kim, Oh, Kim, Ning, Lee, Cha, Tufano and Russell.)
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- 2023
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75. Reducing the inherent auto-inhibitory interaction within the pegRNA enhances prime editing efficiency.
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Ponnienselvan K, Liu P, Nyalile T, Oikemus S, Maitland SA, Lawson ND, Luban J, and Wolfe SA
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- Animals, Humans, Binding Sites, Cold-Shock Response, CRISPR-Cas Systems, Mammals, Ribonucleoproteins, Cold Temperature, Gene Editing, Zebrafish genetics
- Abstract
Prime editing systems have enabled the incorporation of precise edits within a genome without introducing double strand breaks. Previous studies defined an optimal primer binding site (PBS) length for the pegRNA of ∼13 nucleotides depending on the sequence composition. However, optimal PBS length characterization has been based on prime editing outcomes using plasmid or lentiviral expression systems. In this study, we demonstrate that for prime editor (PE) ribonucleoprotein complexes, the auto-inhibitory interaction between the PBS and the spacer sequence affects pegRNA binding efficiency and target recognition. Destabilizing this auto-inhibitory interaction by reducing the complementarity between the PBS-spacer region enhances prime editing efficiency in multiple prime editing formats. In the case of end-protected pegRNAs, a shorter PBS length with a PBS-target strand melting temperature near 37°C is optimal in mammalian cells. Additionally, a transient cold shock treatment of the cells post PE-pegRNA delivery further increases prime editing outcomes for pegRNAs with optimized PBS lengths. Finally, we show that prime editor ribonucleoprotein complexes programmed with pegRNAs designed using these refined parameters efficiently correct disease-related genetic mutations in patient-derived fibroblasts and efficiently install precise edits in primary human T cells and zebrafish., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)
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- 2023
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76. Genome-wide profiling of prime editor off-target sites in vitro and in vivo using PE-tag.
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Liang SQ, Liu P, Ponnienselvan K, Suresh S, Chen Z, Kramme C, Chatterjee P, Zhu LJ, Sontheimer EJ, Xue W, and Wolfe SA
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- Mice, Animals, DNA genetics, DNA Breaks, Double-Stranded, Cell Line, CRISPR-Cas Systems, Mammals genetics, Gene Editing methods, Genome
- Abstract
Prime editors have a broad range of potential research and clinical applications. However, methods to delineate their genome-wide editing activities have generally relied on indirect genome-wide editing assessments or the computational prediction of near-cognate sequences. Here we describe a genome-wide approach for the identification of potential prime editor off-target sites, which we call PE-tag. This method relies on the attachment or insertion of an amplification tag at sites of prime editor activity to allow their identification. PE-tag enables genome-wide profiling of off-target sites in vitro using extracted genomic DNA, in mammalian cell lines and in the adult mouse liver. PE-tag components can be delivered in a variety of formats for off-target site detection. Our studies are consistent with the high specificity previously described for prime editor systems, but we find that off-target editing rates are influenced by prime editing guide RNA design. PE-tag represents an accessible, rapid and sensitive approach for the genome-wide identification of prime editor activity and the evaluation of prime editor safety., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)
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- 2023
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77. Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells.
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Zeng J, Nguyen MA, Liu P, Ferreira da Silva L, Lin LY, Justus DG, Petri K, Clement K, Porter SN, Verma A, Neri NR, Rosanwo T, Ciuculescu MF, Abriss D, Mintzer E, Maitland SA, Demirci S, Tisdale JF, Williams DA, Zhu LJ, Pruett-Miller SM, Pinello L, Joung JK, Pattanayak V, Manis JP, Armant M, Pellin D, Brendel C, Wolfe SA, and Bauer DE
- Abstract
Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on- target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs.
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- 2023
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78. Pre-existing immunity does not impair the engraftment of CRISPR-Cas9-edited cells in rhesus macaques conditioned with busulfan or radiation.
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Essawi K, Hakami W, Naeem Khan MB, Martin R, Zeng J, Chu R, Uchida N, Bonifacino AC, Krouse AE, Linde NS, Donahue RE, Blobel GA, Gerdemann U, Kean LS, Maitland SA, Wolfe SA, Metais JY, Gottschalk S, Bauer DE, Tisdale JF, and Demirci S
- Abstract
CRISPR-Cas9-based therapeutic genome editing approaches hold promise to cure a variety of human diseases. Recent findings demonstrate pre-existing immunity for the commonly used Cas orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans, which threatens the success of this powerful tool in clinical use. Thus, a comprehensive investigation and potential risk assessment are required to exploit the full potential of the system. Here, we investigated existence of immunity to SpCas9 and SaCas9 in control rhesus macaques ( Macaca mulatta) alongside monkeys transplanted with either lentiviral transduced or CRISPR-SpCas9 ribonucleoprotein (RNP)-edited cells. We observed significant levels of Cas9 antibodies in the peripheral blood of all transplanted and non-transplanted control animals. Transplantation of ex vivo transduced or SpCas9-mediated BCL11A enhancer-edited cells did not alter the levels of Cas9 antibodies in rhesus monkeys. Following stimulation of peripheral blood cells with SpCas9 or SaCas9, neither Cas9-specific T cells nor cytokine induction were detected. Robust and durable editing frequencies and expression of high levels of fetal hemoglobin in BCL11A enhancer-edited rhesus monkeys with no evidence of an immune response (>3 years) provide an optimistic outlook for the use of ex vivo CRISPR-SpCas9 (RNP)-edited cells., Competing Interests: S.G. has patent applications in the fields of cell and/or gene therapy for cancer. S.G. is a consultant of TESSA Therapeutics, a DSMB member of Immatics, and has received honoraria from Tidal, Catamaran Bio, Sanofi, and Novartis within the last 2 years.
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- 2023
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79. Self-delivering CRISPR RNAs for AAV Co-delivery and Genome Editing in vivo .
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Zhang H, Kelly K, Lee J, Echeverria D, Cooper D, Panwala R, Chen Z, Gaston N, Newby GA, Xie J, Liu DR, Gao G, Wolfe SA, Khvorova A, Watts JK, and Sontheimer EJ
- Abstract
Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a "protecting oligo"), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo . Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation., Competing Interests: Declaration of Competing Interests E.J.S., J.K.W, A.K., S.A.W., and H.Z. are co-inventors on patent filings related to this work. G.G. is a scientific co-founder, scientific advisor, and equity holder of Voyager Therapeutics, Adrenas Therapeutics, and Aspa Therapeutics. E.J.S. is a co-founder, scientific advisor, and equity holder of Intellia Therapeutics, and a member of the Scientific Advisory Board (S.A.B.) of Tessera Therapeutics. A.K. is a member of the S.A.B. of Prime Medicine. D.R.L is a consultant and equity owner of Prime Medicine, Beam Therapeutics, Pairwise Plants, Nvelop Therapeutics, and Chroma Medicine, companies that use or deliver genome editing or epigenome-modulating agents. S.A.W. is a consultant for Chroma Medicine and serves on the S.A.B. for Graphite Bio.
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- 2023
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80. Handheld Autofluorescence Imaging System for Intraoperative Parathyroid Detection.
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Nagururu NV, Ali KM, Seo S, Kim Y, Wolfe SA, Cha J, and Russell JO
- Abstract
Introduction: Hypoparathyroidism and hypocalcemia are common complications after thyroid surgery. Parathyroids may be incidentally damaged or removed because they are difficult to distinguish from surrounding tissue. Intraoperative optical technologies such as near infrared autofluorescence (NIRAF) are becoming increasingly popular to help identify parathyroids during thyroid surgery. The objective of this video is to introduce a developing NIRAF device called hANDY-i and compare the device with existing Food and Drug Administration approved technology., Materials and Methods: hANDY-i is developed by Optosurgical, LLC. The device consists of a coaxial 785 nm laser excitation module and coregistred red-green-blue and near-infrared cameras. Operation of the device and output from preliminary intraoperative use are shown., Results: hANDY-i performs well, producing intuitive side-by-side NIRAF and RGB images of the operating field. The device demonstrates high contrast between suspected parathyroid glands and surrounding tissue. Operating theater, overhead lamps, and surgical headlights can all be used with the device. The device is also shown to be effective in both in vivo and ex vivo applications., Conclusions: The prototype described advance NIRAF technology by reducing light sensitivity and improving output representation. In doing so, hANDY-i makes NIRAF more accessible and less obstructive to the surgical workflow., Sources of Funding: This study was supported by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Number R43EB030874., Disclaimer: The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Yoseph Kim is an employee of Optosurgical LLC. Jaepyeong Cha has ownership stake in Optosurgical LLC. For all other authors, no competing financial interests exist.Runtime of video: 7 mins 14 secs., (Copyright 2023, Mary Ann Liebert, Inc. and American Thyroid Association.)
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- 2023
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81. Human genetic diversity alters off-target outcomes of therapeutic gene editing.
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Cancellieri S, Zeng J, Lin LY, Tognon M, Nguyen MA, Lin J, Bombieri N, Maitland SA, Ciuculescu MF, Katta V, Tsai SQ, Armant M, Wolfe SA, Giugno R, Bauer DE, and Pinello L
- Subjects
- Humans, Hematopoietic Stem Cells, INDEL Mutation, RNA, Guide, CRISPR-Cas Systems, Gene Editing methods, CRISPR-Cas Systems genetics
- Abstract
CRISPR gene editing holds great promise to modify DNA sequences in somatic cells to treat disease. However, standard computational and biochemical methods to predict off-target potential focus on reference genomes. We developed an efficient tool called CRISPRme that considers single-nucleotide polymorphism (SNP) and indel genetic variants to nominate and prioritize off-target sites. We tested the software with a BCL11A enhancer targeting guide RNA (gRNA) showing promise in clinical trials for sickle cell disease and β-thalassemia and found that the top candidate off-target is produced by an allele common in African-ancestry populations (MAF 4.5%) that introduces a protospacer adjacent motif (PAM) sequence. We validated that SpCas9 generates strictly allele-specific indels and pericentric inversions in CD34
+ hematopoietic stem and progenitor cells (HSPCs), although high-fidelity Cas9 mitigates this off-target. This report illustrates how genetic variants should be considered as modifiers of gene editing outcomes. We expect that variant-aware off-target assessment will become integral to therapeutic genome editing evaluation and provide a powerful approach for comprehensive off-target nomination., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)- Published
- 2023
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82. Functional restoration of mouse Nf1 nonsense alleles in differentiated cultured neurons.
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Wu C, Iyer S, Wolfe SA, and Jacobson A
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- Animals, Mice, Genes, Neurofibromatosis 1, Codon, Nonsense genetics, Alleles, Mutation, Neurons, Codon, Neurofibromin 1 genetics, Neurofibromatosis 1 genetics
- Abstract
Neurofibromatosis type 1 (NF1), one of the most common autosomal dominant genetic disorders, is caused by mutations in the NF1 gene. NF1 patients have a wide variety of manifestations with a subset at high risk for the development of tumors in the central nervous system (CNS). Nonsense mutations that result in the synthesis of truncated NF1 protein (neurofibromin) are strongly associated with CNS tumors. Therapeutic nonsense suppression with small molecule drugs is a potentially powerful approach to restore the expression of genes harboring nonsense mutations. Ataluren is one such drug that has been shown to restore full-length functional protein in several models of nonsense mutation diseases, as well as in patients with nonsense mutation Duchenne muscular dystrophy. To test ataluren's potential applicability to NF1 nonsense mutations associated with CNS tumors, we generated a homozygous Nf1
R683X/R683X -3X-FLAG mouse embryonic stem (mES) cell line which recapitulates an NF1 patient nonsense mutation (c.2041 C > T; p.Arg681X). We differentiated Nf1R683X/R683X -3X-FLAG mES cells into cortical neurons in vitro, treated the cells with ataluren, and demonstrated that ataluren can promote readthrough of the nonsense mutation at codon 683 of Nf1 mRNA in neural cells. The resulting full-length protein is able to reduce the cellular level of hyperactive phosphorylated ERK (pERK), a RAS effector normally suppressed by the NF1 protein., (© 2022. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)- Published
- 2022
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83. Efficient Homology-Directed Repair with Circular Single-Stranded DNA Donors.
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Iyer S, Mir A, Vega-Badillo J, Roscoe BP, Ibraheim R, Zhu LJ, Lee J, Liu P, Luk K, Mintzer E, Guo D, Soares de Brito J, Emerson CP Jr, Zamore PD, Sontheimer EJ, and Wolfe SA
- Subjects
- Humans, CRISPR-Cas Systems genetics, DNA metabolism, Endonucleases genetics, HEK293 Cells, K562 Cells, DNA, Single-Stranded genetics, Gene Editing methods
- Abstract
While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. In this study, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified traffic light reporter (TLR) system (TLR-multi-Cas variant 1 [MCV1]) that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knockins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knockins in mammalian cell lines.
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- 2022
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84. Invited Discussion on: "Risk Factors Analysis for Different Types of Unfavorable Fracture Patterns During Sagittal Split Ramus Osteotomy-A Retrospective Study of 2008 Sides".
- Author
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Wolfe SA, Piccinini PS, and Wolfe E
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- Humans, Retrospective Studies, Mandible, Risk Factors, Osteotomy, Sagittal Split Ramus adverse effects, Fractures, Bone
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- 2022
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85. Ethanol withdrawal-induced adaptations in prefrontal corticotropin releasing factor receptor 1-expressing neurons regulate anxiety and conditioned rewarding effects of ethanol.
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Patel RR, Wolfe SA, Borgonetti V, Gandhi PJ, Rodriguez L, Snyder AE, D'Ambrosio S, Bajo M, Domissy A, Head S, Contet C, Dayne Mayfield R, Roberts AJ, and Roberto M
- Subjects
- Humans, Receptors, Corticotropin-Releasing Hormone genetics, Ethanol pharmacology, Corticotropin-Releasing Hormone, Neurons, Anxiety, Alcoholism genetics, Substance Withdrawal Syndrome
- Abstract
Prefrontal circuits are thought to underlie aberrant emotion contributing to relapse in abstinence; however, the discrete cell-types and mechanisms remain largely unknown. Corticotropin-releasing factor and its cognate type-1 receptor, a prominent brain stress system, is implicated in anxiety and alcohol use disorder (AUD). Here, we tested the hypothesis that medial prefrontal cortex CRF1-expressing (mPFC
CRF1+ ) neurons comprise a distinct population that exhibits neuroadaptations following withdrawal from chronic ethanol underlying AUD-related behavior. We found that mPFCCRF1+ neurons comprise a glutamatergic population with distinct electrophysiological properties and regulate anxiety and conditioned rewarding effects of ethanol. Notably, mPFCCRF1+ neurons undergo unique neuroadaptations compared to neighboring neurons including a remarkable decrease in excitability and glutamatergic signaling selectively in withdrawal, which is driven in part by the basolateral amygdala. To gain mechanistic insight into these electrophysiological adaptations, we sequenced the transcriptome of mPFCCRF1+ neurons and found that withdrawal leads to an increase in colony-stimulating factor 1 (CSF1) in this population. We found that selective overexpression of CSF1 in mPFCCRF1+ neurons is sufficient to decrease glutamate transmission, heighten anxiety, and abolish ethanol reinforcement, providing mechanistic insight into the observed mPFCCRF1+ synaptic adaptations in withdrawal that drive these behavioral phenotypes. Together, these findings highlight mPFCCRF1+ neurons as a critical site of enduring adaptations that may contribute to the persistent vulnerability to ethanol misuse in abstinence, and CSF1 as a novel target for therapeutic intervention for withdrawal-related negative affect., (© 2022. The Author(s).)- Published
- 2022
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86. Alcohol Dependence Induces CRF Sensitivity in Female Central Amygdala GABA Synapses.
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Rodriguez L, Kirson D, Wolfe SA, Patel RR, Varodayan FP, Snyder AE, Gandhi PJ, Khom S, Vlkolinsky R, Bajo M, and Roberto M
- Subjects
- Animals, Corticotropin-Releasing Hormone metabolism, Ethanol pharmacology, Female, Humans, Male, Rats, Receptors, Corticotropin-Releasing Hormone genetics, Receptors, Corticotropin-Releasing Hormone metabolism, Synapses metabolism, Synaptic Transmission, gamma-Aminobutyric Acid pharmacology, Alcoholism, Central Amygdaloid Nucleus metabolism
- Abstract
Alcohol use disorder (AUD) is a chronically relapsing disease characterized by loss of control in seeking and consuming alcohol (ethanol) driven by the recruitment of brain stress systems. However, AUD differs among the sexes: men are more likely to develop AUD, but women progress from casual to binge drinking and heavy alcohol use more quickly. The central amygdala (CeA) is a hub of stress and anxiety, with corticotropin-releasing factor (CRF)-CRF
1 receptor and Gamma-Aminobutyric Acid (GABA)-ergic signaling dysregulation occurring in alcohol-dependent male rodents. However, we recently showed that GABAergic synapses in female rats are less sensitive to the acute effects of ethanol. Here, we used patch-clamp electrophysiology to examine the effects of alcohol dependence on the CRF modulation of rat CeA GABAergic transmission of both sexes. We found that GABAergic synapses of naïve female rats were unresponsive to CRF application compared to males, although alcohol dependence induced a similar CRF responsivity in both sexes. In situ hybridization revealed that females had fewer CeA neurons containing mRNA for the CRF1 receptor ( Crhr1 ) than males, but in dependence, the percentage of Crhr1 -expressing neurons in females increased, unlike in males. Overall, our data provide evidence for sexually dimorphic CeA CRF system effects on GABAergic synapses in dependence.- Published
- 2022
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87. Proliferation of steatocystomas in 2 transgender men.
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Porras Fimbres DC, Wolfe SA, and Kelley CE
- Abstract
Competing Interests: None disclosed.
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- 2022
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88. The Amygdala Noradrenergic System Is Compromised With Alcohol Use Disorder.
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Varodayan FP, Patel RR, Matzeu A, Wolfe SA, Curley DE, Khom S, Gandhi PJ, Rodriguez L, Bajo M, D'Ambrosio S, Sun H, Kerr TM, Gonzales RA, Leggio L, Natividad LA, Haass-Koffler CL, Martin-Fardon R, and Roberto M
- Subjects
- Alcohol Drinking, Animals, Ethanol pharmacology, Humans, Male, Norepinephrine, RNA, Messenger, Rats, Receptors, Adrenergic metabolism, Alcoholism, Central Amygdaloid Nucleus metabolism
- Abstract
Background: Alcohol use disorder (AUD) is a leading preventable cause of death. The central amygdala (CeA) is a hub for stress and AUD, while dysfunction of the noradrenaline stress system is implicated in AUD relapse., Methods: Here, we investigated whether alcohol (ethanol) dependence and protracted withdrawal alter noradrenergic regulation of the amygdala in rodents and humans. Male adult rats were housed under control conditions, subjected to chronic intermittent ethanol vapor exposure to induce dependence, or withdrawn from chronic intermittent ethanol vapor exposure for 2 weeks, and ex vivo electrophysiology, biochemistry (catecholamine quantification by high-performance liquid chromatography), in situ hybridization, and behavioral brain-site specific pharmacology studies were performed. We also used real-time quantitative polymerase chain reaction to assess gene expression of α
1 B , β1 , and β2 adrenergic receptors in human postmortem brain tissue from men diagnosed with AUD and matched control subjects., Results: We found that α1 receptors potentiate CeA GABAergic (gamma-aminobutyric acidergic) transmission and drive moderate alcohol intake in control rats. In dependent rats, β receptors disinhibit a subpopulation of CeA neurons, contributing to their excessive drinking. Withdrawal produces CeA functional recovery with no change in local noradrenaline tissue concentrations, although there are some long-lasting differences in the cellular patterns of adrenergic receptor messenger RNA expression. In addition, postmortem brain analyses reveal increased α1 B receptor messenger RNA in the amygdala of humans with AUD., Conclusions: CeA adrenergic receptors are key neural substrates of AUD. Identification of these novel mechanisms that drive alcohol drinking, particularly during the alcohol-dependent state, supports ongoing new medication development for AUD., (Copyright © 2022 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)- Published
- 2022
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89. Adenine Base Editing In Vivo with a Single Adeno-Associated Virus Vector.
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Zhang H, Bamidele N, Liu P, Ojelabi O, Gao XD, Rodriguez T, Cheng H, Kelly K, Watts JK, Xie J, Gao G, Wolfe SA, Xue W, and Sontheimer EJ
- Abstract
Base editors (BEs) have opened new avenues for the treatment of genetic diseases. However, advances in delivery approaches are needed to enable disease targeting of a broad range of tissues and cell types. Adeno-associated virus (AAV) vectors remain one of the most promising delivery vehicles for gene therapies. Currently, most BE/guide combinations and their promoters exceed the packaging limit (∼5 kb) of AAVs. Dual-AAV delivery strategies often require high viral doses that impose safety concerns. In this study, we engineered an adenine base editor (ABE) using a compact Cas9 from Neisseria meningitidis (Nme2Cas9). Compared with the well-characterized Streptococcus pyogenes Cas9-containing ABEs, ABEs using Nme2Cas9 (Nme2-ABE) possess a distinct protospacer adjacent motif (N
4 CC) and editing window, exhibit fewer off-target effects, and can efficiently install therapeutically relevant mutations in both human and mouse genomes. Importantly, we show that in vivo delivery of Nme2-ABE and its guide RNA by a single AAV vector can efficiently edit mouse genomic loci and revert the disease mutation and phenotype in an adult mouse model of tyrosinemia. We anticipate that Nme2-ABE, by virtue of its compact size and broad targeting range, will enable a range of therapeutic applications with improved safety and efficacy due in part to packaging in a single-vector system., Competing Interests: H.Z., N.B., P.L., X.D.G., S.A.W., W.X., and E.J.S. are coinventors on patent filings related to this study. G.G. is scientific cofounder, scientific advisor, and equity holder of Voyager Therapeutics, Adrenas Therapeutics, and Aspa Therapeutics. E.J.S. is a cofounder, scientific advisor, and equity holder of Intellia Therapeutics, and a member of the scientific advisory board of Tessera Therapeutics., (Copyright 2022, Mary Ann Liebert, Inc., publishers.)- Published
- 2022
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90. Optimization of Nuclear Localization Signal Composition Improves CRISPR-Cas12a Editing Rates in Human Primary Cells.
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Luk K, Liu P, Zeng J, Wang Y, Maitland SA, Idrizi F, Ponnienselvan K, Iyer S, Zhu LJ, Luban J, Bauer DE, and Wolfe SA
- Abstract
Type V CRISPR-Cas12a systems are an attractive Cas9-alternative nuclease platform for specific genome editing applications. However, previous studies demonstrate that there is a gap in overall activity between Cas12a and Cas9 in primary cells.
1 Here we describe optimization to the NLS composition and architecture of Cas12a to facilitate highly efficient targeted mutagenesis in human transformed cell lines (HEK293T, Jurkat, and K562 cells) and primary cells (NK cells and CD34+ HSPCs), regardless of Cas12a ortholog. Our 3xNLS Cas12a architecture resulted in the most robust editing platform. The improved editing activity of Cas12a in both NK cells and CD34+ HSPCs resulted in pronounced phenotypic changes associated with target gene editing. Lastly, we demonstrated that optimization of the NLS composition and architecture of Cas12a did not increase editing at potential off-target sites in HEK293T or CD34+ HSPCs. Our new Cas12a NLS variant provides an improved nuclease platform for therapeutic genome editing., Competing Interests: Author disclosure statement The authors declare the following competing interests: The authors have filed patent applications related to Cas12a variants; D.E.B. and J.Z. has patents related to BCL11A enhancer genome editing; all other authors have no competing interests.- Published
- 2022
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91. A flexible split prime editor using truncated reverse transcriptase improves dual-AAV delivery in mouse liver.
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Zheng C, Liang SQ, Liu B, Liu P, Kwan SY, Wolfe SA, and Xue W
- Subjects
- Animals, Liver, Mammals, Mice, RNA-Directed DNA Polymerase, Ribonuclease H, Gene Editing, Proprotein Convertase 9 genetics
- Abstract
Prime editor (PE) has tremendous promise for gene therapy. However, it remains a challenge to deliver PE (>6.3 kb) in vivo. Although PE can be split into two fragments and delivered using dual adeno-associated viruses (AAVs), choice of split sites within Cas9-which affects editing efficiency-is limited due to the large size of PE. Furthermore, overexpressing reverse transcriptase in mammalian cells might disrupt translation termination via its RNase H domain. Here, we developed a compact PE without the RNase H domain that showed editing comparable with full-length PE. With compact PE, we used a Cas9 split site (Glu 573) that supported robust editing in cells (up to 93% of full-length PE) and in mouse liver. We then demonstrated that split-cPE573 delivered by dual-AAV8 efficiently mediated a 3-bp TGA insertion in the Pcsk9 gene in mouse liver. Compact PE without the RNase H domain abolished its binding to peptidyl release factor 1 (eRF1) and mitigated the stop codon readthrough effect observed with full-length PE. This study identifies a compact PE with a flexible split design to advance utility of prime editing in vivo., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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92. LONP-1 and ATFS-1 sustain deleterious heteroplasmy by promoting mtDNA replication in dysfunctional mitochondria.
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Yang Q, Liu P, Anderson NS, Shpilka T, Du Y, Naresh NU, Li R, Zhu LJ, Luk K, Lavelle J, Zeinert RD, Chien P, Wolfe SA, and Haynes CM
- Subjects
- Animals, Humans, Animals, Genetically Modified, Cell Line, DNA Polymerase gamma genetics, DNA Polymerase gamma metabolism, Proteolysis, ATP-Dependent Proteases genetics, ATP-Dependent Proteases metabolism, Caenorhabditis elegans genetics, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins genetics, Caenorhabditis elegans Proteins metabolism, DNA Replication, DNA, Mitochondrial biosynthesis, DNA, Mitochondrial genetics, Heteroplasmy, Mitochondria genetics, Mitochondria metabolism, Mitochondria pathology, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Oxidative Phosphorylation, Transcription Factors genetics, Transcription Factors metabolism
- Abstract
The accumulation of deleterious mitochondrial DNA (∆mtDNA) causes inherited mitochondrial diseases and ageing-associated decline in mitochondrial functions such as oxidative phosphorylation. Following mitochondrial perturbations, the bZIP protein ATFS-1 induces a transcriptional programme to restore mitochondrial function. Paradoxically, ATFS-1 is also required to maintain ∆mtDNAs in heteroplasmic worms. The mechanism by which ATFS-1 promotes ∆mtDNA accumulation relative to wild-type mtDNAs is unclear. Here we show that ATFS-1 accumulates in dysfunctional mitochondria. ATFS-1 is absent in healthy mitochondria owing to degradation by the mtDNA-bound protease LONP-1, which results in the nearly exclusive association between ATFS-1 and ∆mtDNAs in heteroplasmic worms. Moreover, we demonstrate that mitochondrial ATFS-1 promotes the binding of the mtDNA replicative polymerase (POLG) to ∆mtDNAs. Interestingly, inhibition of the mtDNA-bound protease LONP-1 increased ATFS-1 and POLG binding to wild-type mtDNAs. LONP-1 inhibition in Caenorhabditis elegans and human cybrid cells improved the heteroplasmy ratio and restored oxidative phosphorylation. Our findings suggest that ATFS-1 promotes mtDNA replication in dysfunctional mitochondria by promoting POLG-mtDNA binding, which is antagonized by LONP-1., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)
- Published
- 2022
- Full Text
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93. Invited Discussion on: Oral and Maxillofacial Autologous Fat Transplantation: History, Clinical Application Status and Research Progress.
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Wolfe SA
- Subjects
- Humans, Transplantation, Autologous, Adipose Tissue
- Published
- 2022
- Full Text
- View/download PDF
94. The Synaptic Interactions of Alcohol and the Endogenous Cannabinoid System.
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Wolfe SA, Vozella V, and Roberto M
- Subjects
- Endocannabinoids pharmacology, Ethanol pharmacology, Humans, Receptors, Cannabinoid, Synaptic Transmission, Alcoholism, Cannabinoids pharmacology
- Abstract
Purpose: A growing body of evidence has implicated the endocannabinoid (eCB) system in the acute, chronic, and withdrawal effects of alcohol/ethanol on synaptic function. These eCB-mediated synaptic effects may contribute to the development of alcohol use disorder (AUD). Alcohol exposure causes neurobiological alterations similar to those elicited by chronic cannabinoid (CB) exposure. Like alcohol, cannabinoids alter many central processes, such as cognition, locomotion, synaptic transmission, and neurotransmitter release. There is a strong need to elucidate the effects of ethanol on the eCB system in different brain regions to understand the role of eCB signaling in AUD., Search Methods: For the scope of this review, preclinical studies were identified through queries of the PubMed database., Search Results: This search yielded 459 articles. Clinical studies and papers irrelevant to the topic of this review were excluded., Discussion and Conclusions: The endocannabinoid system includes, but is not limited to, cannabinoid receptors 1 (CB
1 ), among the most abundantly expressed neuronal receptors in the brain; cannabinoid receptors 2 (CB2 ); and endogenously formed CB1 ligands, including arachidonoylethanolamide (AEA; anandamide), and 2-arachidonoylglycerol (2-AG). The development of specific CB1 agonists, such as WIN 55,212-2 (WIN), and antagonists, such as SR 141716A (rimonabant), provide powerful pharmacological tools for eCB research. Alcohol exposure has brain region-specific effects on the eCB system, including altering the synthesis of endocannabinoids (e.g., AEA, 2-AG), the synthesis of their precursors, and the density and coupling efficacy of CB1 . These alcohol-induced alterations of the eCB system have subsequent effects on synaptic function including neuronal excitability and postsynaptic conductance. This review will provide a comprehensive evaluation of the current literature on the synaptic interactions of alcohol exposure and eCB signaling systems, with an emphasis on molecular and physiological synaptic effects of alcohol on the eCB system. A limited volume of studies has focused on the underlying interactions of alcohol and the eCB system at the synaptic level in the brain. Thus, the data on synaptic interactions are sparse, and future research addressing these interactions is much needed., Competing Interests: Disclosures Dr. Roberto is Neuropharmacology senior section editor. The authors declare no competing financial or nonfinancial interests.- Published
- 2022
- Full Text
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95. Genome-wide detection of CRISPR editing in vivo using GUIDE-tag.
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Liang SQ, Liu P, Smith JL, Mintzer E, Maitland S, Dong X, Yang Q, Lee J, Haynes CM, Zhu LJ, Watts JK, Sontheimer EJ, Wolfe SA, and Xue W
- Subjects
- Animals, Base Sequence, Biotin metabolism, Biotinylation, CRISPR-Associated Protein 9 metabolism, Cell Line, Tumor, DNA metabolism, Genes, Reporter, Genome, Liver metabolism, Lung metabolism, Mice, Ribonucleoproteins metabolism, CRISPR-Cas Systems genetics, Gene Editing, RNA, Guide, CRISPR-Cas Systems genetics
- Abstract
Analysis of off-target editing is an important aspect of the development of safe nuclease-based genome editing therapeutics. in vivo assessment of nuclease off-target activity has primarily been indirect (based on discovery in vitro, in cells or via computational prediction) or through ChIP-based detection of double-strand break (DSB) DNA repair factors, which can be cumbersome. Herein we describe GUIDE-tag, which enables one-step, off-target genome editing analysis in mouse liver and lung. The GUIDE-tag system utilizes tethering between the Cas9 nuclease and the DNA donor to increase the capture rate of nuclease-mediated DSBs and UMI incorporation via Tn5 tagmentation to avoid PCR bias. These components can be delivered as SpyCas9-mSA ribonucleoprotein complexes and biotin-dsDNA donor for in vivo editing analysis. GUIDE-tag enables detection of off-target sites where editing rates are ≥ 0.2%. UDiTaS analysis utilizing the same tagmented genomic DNA detects low frequency translocation events with off-target sites and large deletions in vivo. The SpyCas9-mSA and biotin-dsDNA system provides a method to capture DSB loci in vivo in a variety of tissues with a workflow that is amenable to analysis of gross genomic alterations that are associated with genome editing., (© 2022. The Author(s).)
- Published
- 2022
- Full Text
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96. Commentary on: The Aging Surgeon: Evidence and Experience.
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Wolfe SA and Wolfe EM
- Subjects
- Aging, Humans, Surgeons
- Published
- 2022
- Full Text
- View/download PDF
97. Parathyroid Adenoma
- Author
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Wolfe SA and Sharma S
- Abstract
The parathyroid glands were first identified in animals in the mid-1880s. Felix Mandl performed the first parathyroidectomy in Vienna in 1925. Since then, there have been many advances in the understanding, diagnosis, and treatment of parathyroid disease. The parathyroid glands are small, oval-shaped structures found close to the thyroid. Eighty-five percent of patients have 4 glands, with 2 lying superior and 2 inferior. The superior glands (developed from the fourth branchial pouch) are usually located on the posterior-lateral surface of the middle to superior thyroid lobe. The inferior glands are more variable in location due to their embryological descent with the thymus (both developing from the third branchial pouch) and can be found at any point along its path of descent. Most commonly, they are located in the inferior third of the thyroid gland. A normal parathyroid gland is the size of an apple seed and weighs approximately 0.5 g. Microadenomas are defined as tumors that weigh less than 0.1 g, while a giant adenoma is over 2 g. The average weight of an adenoma is 1 g., (Copyright © 2022, StatPearls Publishing LLC.)
- Published
- 2022
98. Allele-Specific Knockdown of Mutant Huntingtin Protein via Editing at Coding Region Single Nucleotide Polymorphism Heterozygosities.
- Author
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Oikemus SR, Pfister EL, Sapp E, Chase KO, Kennington LA, Hudgens E, Miller R, Zhu LJ, Chaudhary A, Mick EO, Sena-Esteves M, Wolfe SA, DiFiglia M, Aronin N, and Brodsky MH
- Subjects
- Alleles, Animals, Huntingtin Protein genetics, Mice, Polymorphism, Single Nucleotide, Huntington Disease genetics, Huntington Disease therapy
- Abstract
Huntington's disease (HD) is a devastating, autosomal dominant neurodegenerative disease caused by a trinucleotide repeat expansion in the huntingtin (HTT) gene. Inactivation of the mutant allele by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 based gene editing offers a possible therapeutic approach for this disease, but permanent disruption of normal HTT function might compromise adult neuronal function. Here, we use a novel HD mouse model to examine allele-specific editing of mutant HTT (mHTT), with a BAC97 transgene expressing mHTT and a YAC18 transgene expressing normal HTT. We achieve allele-specific inactivation of HTT by targeting a protein coding sequence containing a common, heterozygous single nucleotide polymorphism (SNP). The outcome is a marked and allele-selective reduction of mHTT protein in a mouse model of HD. Expression of a single CRISPR-Cas9 nuclease in neurons generated a high frequency of mutations in the targeted HD allele that included both small insertion/deletion (InDel) mutations and viral vector insertions. Thus, allele-specific targeting of InDel and insertion mutations to heterozygous coding region SNPs provides a feasible approach to inactivate autosomal dominant mutations that cause genetic disease.
- Published
- 2022
- Full Text
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99. Parathyroid Minimally Invasive Surgery
- Author
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Wolfe SA and Fingeret A
- Abstract
Historically, a bilateral exploration was performed for the treatment of hyperparathyroidism. However, in 85% of cases of hyperparathyroidism, a single adenoma is the cause. With the advent of increasingly accurate localization studies, a minimally invasive approach to parathyroid surgery is emerging as a standard of care., (Copyright © 2022, StatPearls Publishing LLC.)
- Published
- 2022
100. Gingivosupraperiosteoplasty following Presurgical Maxillary Orthopedics Is Associated with Normal Midface Growth in Complete Unilateral and Bilateral Cleft Patients at Mixed Dentition.
- Author
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Mejia M, Wolfe EM, Murphy BD, Rothenberg L, Tejero A, Bauer M, and Wolfe SA
- Subjects
- Cephalometry, Cleft Lip complications, Cleft Palate complications, Dentition, Mixed, Face anatomy & histology, Female, Humans, Infant, Infant, Newborn, Male, Maxilla growth & development, Maxilla surgery, Orthodontic Appliances, Periosteum surgery, Treatment Outcome, Cleft Lip therapy, Cleft Palate surgery, Gingivoplasty methods, Maxillofacial Development, Palatal Obturators
- Abstract
Background: Passive orthodontic appliances and gingivosupraperiosteoplasty are adjuncts that can be used by surgeons at the time of primary cleft lip repair. These treatments, along with the surgical technique of cleft lip and palate repair, may impact midface growth. The objective of this study was to describe the authors' protocol for unilateral and bilateral cleft lip repair and to evaluate midfacial growth in a cohort of patients at mixed dentition who had undergone presurgical passive orthodontic appliance therapy and gingivosupraperiosteoplasty at the time of unilateral and bilateral cleft lip repair., Methods: Fifteen complete unilateral and 15 complete bilateral cleft lip and palate patients underwent passive orthodontic appliance treatment and primary lip repair with gingivosupraperiosteoplasty. Lateral cephalograms were analyzed by three blinded reviewers. Mean cephalometric measurements at mixed dentition were compared to cephalometric values for noncleft patients, unilateral cleft lip and palate patients who did not undergo gingivoperiosteoplasty or presurgical treatment, and unilateral cleft lip and palate patients who underwent gingivoperiosteoplasty/nasoalveolar molding with independent samples t tests., Results: Mean cephalometric values were within age-specific normal values for sella-nasion-A point, sella-nasion-B point, A point-nasion-B point, and facial axis. Eighty-seven (13/15) percent of unilateral cleft lip and palate patients and 93 percent (14/15) of bilateral cleft lip and palate patients did not exhibit skeletal class III malocclusion. There was no significant difference between cephalometric values for our patients and patients who did not receive gingivosupraperiosteoplasty or presurgical treatment or who underwent the gingivoperiosteoplasty/nasoalveolar molding protocol., Conclusions: Presurgical passive orthodontic appliances, combined with gingivosupraperiosteoplasty at the time of lip repair, leads to normal maxillary development in most patients at mixed dentition. Assessment of midface growth at skeletal maturity is required., Clinical Question/level of Evidence: Therapeutic, IV., (Copyright © 2021 by the American Society of Plastic Surgeons.)
- Published
- 2021
- Full Text
- View/download PDF
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