Jenny C. Barker

Gene correction by homologous recombination with zinc finger nucleases in primary cells from a mouse model of a generic recessive genetic disease.

Zinc Finger nucleases (ZFNs) have been used to create precise genome modifications at frequencies that might be therapeutically useful in gene therapy. We created a mouse model of a generic recessive genetic disease to establish a preclinical system to develop the use of ZFN-mediated gene correction for gene therapy. We knocked a mutated GFP gene into the ROSA26 locus in murine embryonic stem (ES) cells and used these cells to create a transgenic mouse. We used ZFNs to determine the frequency of gene correction by gene targeting in different primary cells from this model. We achieved targeting frequencies from 0.17 to 6% in different cell types, including primary fibroblasts and astrocytes. We demonstrate that ex vivo gene-corrected fibroblasts can be transplanted back into a mouse where they retained the corrected phenotype. In addition, we achieved targeting frequencies of over 1% in ES cells, and the targeted ES cells retained the ability to differentiate into cell types from all three germline lineages. In summary, potentially therapeutically relevant frequencies of ZFN-mediated gene targeting can be achieved in a variety of primary cells and these cells can then be transplanted back into a recipient.

A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype

BackgroundThe ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Adeno-associated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. There are now a number of natural for that designed AAV serotypes that each has a differential ability to infect a variety of cell types. Although transduction studies have been completed, the bulk of the studies have been done in vivo, and there has never been a comprehensive study of transduction ex vivo/in vitro.MethodsEach cell type was infected with each serotype at a multiplicity of infection of 100,000 viral genomes/cell and transduction was analyzed by flow cytometry + .ResultsWe found that AAV1 and AAV6 have the greatest ability to transduce a wide range of cell types, however, for particular cell types, there are specific serotypes that provide optimal transduction.ConclusionsIn this work, we describe the transduction efficiency of ten different AAV serotypes in thirty-four different mammalian cell lines and primary cell types. Although these results may not be universal due to numerous factors such as, culture conditions and/ or cell growth rates and cell heterogeneity, these results provide an important and unique resource for investigators who use AAV as an ex vivo gene delivery vector or who work with cells that are difficult to transfect.

Simultaneous irrigation and negative pressure wound therapy enhances wound healing and reduces wound bioburden in a porcine model

Infected foot wounds are one of the most common reasons for hospitalization and amputation among persons with diabetes. The objective of the study was to investigate a new wound therapy system that employs negative pressure wound therapy (NPWT) with simultaneous irrigation therapy. For this study, we used a porcine model with full‐thickness excisional wounds, inoculated with Pseudomonas aeruginosa. Wounds were treated for 21 days of therapy with either NPWT, NPWT with simultaneous irrigation therapy using normal saline or polyhexanide biguanide (PHMB) at low or high flow rates, or control. Data show that NPWT with either irrigation condition improved wound healing rates over control‐treated wounds, yet did not differ from NPWT alone. NPWT improved bioburden over control‐treated wounds. NPWT with simultaneous irrigation further reduced bioburden over control and NPWT‐treated wounds; however, flow rate did not affect these outcomes. Together, these data show that NPWT with simultaneous irrigation therapy with either normal saline or PHMB has a positive effect on bioburden in a porcine model, which may translate clinically to improved wound healing outcomes.

Genome Editing in Mouse Spermatogonial Stem/Progenitor Cells Using Engineered Nucleases

Editing the genome to create specific sequence modifications is a powerful way to study gene function and promises future applicability to gene therapy. Creation of precise modifications requires homologous recombination, a very rare event in most cell types that can be stimulated by introducing a double strand break near the target sequence. One method to create a double strand break in a particular sequence is with a custom designed nuclease. We used engineered nucleases to stimulate homologous recombination to correct a mutant gene in mouse “GS” (germline stem) cells, testicular derived cell cultures containing spermatogonial stem cells and progenitor cells. We demonstrated that gene-corrected cells maintained several properties of spermatogonial stem/progenitor cells including the ability to colonize following testicular transplantation. This proof of concept for genome editing in GS cells impacts both cell therapy and basic research given the potential for GS cells to be propagated in vitro, contribute to the germline in vivo following testicular transplantation or become reprogrammed to pluripotency in vitro.

Genome editing of mouse fibroblasts by homologous recombination for sustained secretion of PDGF-B and augmentation of wound healing.

Background: Exogenous cytokines, such as platelet-derived growth factor (PDGF)-B, can augment wound healing, but sustained delivery to maintain therapeutic levels remains a problem. “Genome editing” is a new technology in which precise genome modifications are made within cells using engineered site-specific nucleases. Genome editing avoids many of the complications associated with traditional gene therapy and the use of viral vectors, including random integration, imprecise gene expression, and inadvertent oncogene activation. Methods: This study demonstrates site-specific nuclease-mediated integration of a PDGF-B transgene into a predefined locus within the genome of primary mouse fibroblasts. Engineered fibroblasts were applied to splinted mouse wounds and evaluated after 14 days and 5 months for the retention of engineered fibroblasts, wound healing morphology, angiogenesis, and systemic PDGF-B expression. Results: The application of engineered PDGF-B–expressing fibroblasts enhanced wound healing compared with controls. Low-level, constitutive expression of PDGF-B was achieved without detectable levels of systemic PDGF-B. The mechanism of improved wound healing is, at least in part, the result of increased wound vascularization, as the wounds treated with PDGF-B fibroblasts had a blood vessel density 2.5 times greater than controls. After 5 months, the engineered fibroblasts persisted in the wound bed. No adverse effects were detected from the application of these fibroblasts after 5 months as assessed by hematoxylin and eosin staining of wounds and by mouse necropsy. Conclusions: These data support that site-specific genome editing allows for sustained cell-based cytokine delivery. Furthermore, sustained release of PDGF-B increases the speed and quality of wound healing after a single application.

Current Concepts in Debridement: Science and Strategies

Background: The establishment of a healthy wound bed through adequate debridement of infected, senescent, and/or devitalized tissue is central to the progression of normal wound healing. Although a variety of surgical and nonsurgical strategies have been proposed, none have proven completely effective in all settings. This review focuses on the principles and techniques of modern debridement practices employed in the management of complex wounds. Methods: A comprehensive review of the PubMed/Medline and Ovid databases was performed to identify basic science and clinical studies using key words most relevant to biofilm, debridement, and wound healing. English language articles that were peer reviewed and that met the standard of evidence-based medicine were included. Level of evidence for various debridement approaches was rated utilizing the American Society of Plastic Surgeons Rating Levels of Evidence and Grading Recommendations. Results: The value of both operative and nonoperative debridement techniques, their indications, and limitations are described. With an emphasis placed on surgical debridement, this review highlights technical adjuncts that can be used to optimize wound bed preparation, including preoperative topical staining of the wound, as well as the use of color-guided endpoints to prevent removal of excess healthy tissue. The indications for using temporizing measures for wound control such as negative pressure wound therapy with and without installation are also discussed. Conclusion: Optimal management requires a multimodal approach that centers around operative debridement and incorporates the use of adjunctive measures to facilitate the removal of infected tissue, biofilm, and/or senescent cells that impede the progression of normal wound healing.

Medical Student Mentorship in Plastic Surgery: The Mentee’s Perspective

Background: Mentorship is a universal concept that has a significant impact on nearly every surgical career. Although frequently editorialized, true data investigating the value of mentorship are lacking in the plastic surgery literature. This study evaluates mentorship in plastic surgery from the medical student perspective. Methods: An electronic survey was sent to recently matched postgraduate year–1 integrated track residents in 2014, with a response rate of 76 percent. Results: Seventy-seven percent of students reported a mentoring relationship. Details of the mentoring relationship were defined. Over 80 percent of students reported a mentor’s influence in their decision to pursue plastic surgery, and nearly 40 percent of students expressed interest in practicing the same subspecialty as their mentor. Benefits of the relationship were also described. Mentees value guidance around career preparation and advice and prioritized “a genuine interest in their career and personal development” above all other mentor qualities (p ⩽ 1.6 × 10−16). Mentees prefer frequent, one-on-one interactions over less frequent interaction or group activities. Students did not prefer “assigned” relationships (91 percent), but did prefer “facilitated exposure.” Major barriers to mentorship included mentor time constraints and lack of exposure to plastic surgery. Indeed, significant differences in the presence of a mentoring relationship correlated with involvement of the plastic surgery department in the medical school curriculum. Conclusions: This study defines successes and highlights areas for improvement of mentorship of plastic surgery medical students. Successful mentorship may contribute to the future of plastic surgery, and a commitment toward this endeavor is needed at the local, departmental, and national leadership levels.

Targeted Peripheral Nerve-directed Onabotulinumtoxin A Injection for Effective Long-term Therapy for Migraine Headache.

Background: Onabotulinumtoxin A (BOTOX) is an FDA-approved treatment for chronic migraine headaches (MHs) that involves on-label, high-dose administration across 31 anatomic sites. Anatomically specific peripheral nerve trigger sites have been identified that contribute to MH pathogenesis and are amenable to both BOTOX injection and surgical decompression. These sites do not always correlate with the on-label FDA-approved injection pattern, but represent a more targeted approach. The efficacy of peripheral nerve–directed BOTOX injection as an independent long-term therapeutic option has not been investigated. Methods: The technique for peripheral nerve–directed therapeutic long-term BOTOX injection is described. A retrospective review was subsequently completed for 223 patients with MH. Sixty-six patients elected to proceed with diagnostic BOTOX injections. Of these, 24 continued long-term therapeutic BOTOX injections, whereas 42 matriculated to surgery. Outcomes were tracked. Results: Initial outcomes included significant improvement in migraine headache index (MHI) (53.5 ± 83.0, P < 0.006), headache days/mo (9.2 ± 12.7, P < 0.0009), and migraine severity (2.6 ± 2.5, P < 0.00008) versus baseline. MHI improved from the initiation of diagnostic injections to the establishment of steady-state injections (P < 0.002), and further improved over time (P < 0.05, mean follow-up 615 days) with no desensitization observed. Decompressive surgery resulted in significant improvement in MHI (100.8 ± 109.7, P < 0.0000005), headache days/mo (10.8 ± 12.7, P < 0.000002), migraine severity (3.0 ± 3.8, P < 0.00001), and migraine duration in hours (16.8 ± 21.6, P < 0.0007). MHI improvement with surgery was better than long-term BOTOX injections (P < 0.05). Conclusions: Though inferior to surgical decompression, preliminary data demonstrate that targeted peripheral nerve–directed BOTOX injection is an effective primary therapy for MH representing a possible alternative to nondirected BOTOX injection with decreased dosage requirements and potentially decreased cost.

Nuclease Mediated Targeted Genome Modification in Mammalian Cells

The development of targeted genome modification by using engineered nucleases has been a rapidly developing field in the last few years. In this chapter we review the different types of nucleases that can be engineered for this purpose, the different types of genome modifications that have been created, and examples of targeted genome modification used for both research purposes and for potential gene therapy uses.

A Formidable Foe Is Sabotaging Your Results: What You Should Know about Biofilms and Wound Healing.

Learning Objectives: After reading this article, the participant should be able to: 1. Describe biofilm pathogenesis as it relates to problem wounds. 2. Understand the preclinical and clinical evidence implicating biofilm in problem wounds. 3. Explain the diagnostic and treatment challenges that biofilms create for problem wounds. 4. Demonstrate a basic understanding of emerging strategies aimed at counteracting these processes. Summary: Biofilm represents a protected mode of growth for bacteria, allowing them to evade standard diagnostic techniques and avoid eradication by standard therapies. Although only recently discovered, biofilm has existed for millennia and complicates nearly every aspect of medicine. Biofilm impacts wound healing by allowing bacteria to evade immune responses, prolonging inflammation and disabling skin barrier function. It is important to understand why problem wounds persist despite state-of-the-art treatment, why they are difficult to accurately diagnose, and why they recur. The aim of this article is to focus on current gaps in knowledge related to problem wounds, specifically, biofilm infection.