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1.
Mol Ther Methods Clin Dev ; 29: 227-235, 2023 Jun 08.
Article in English | MEDLINE | ID: mdl-37090476

ABSTRACT

Ocular graft versus host disease (OGvHD) develops after allogeneic hematopoietic stem cell transplantation (HSCT) and manifests as ocular surface inflammatory disease. This study evaluated the efficacy of adeno-associated virus (AAV) gene therapy encoding human leukocyte antigen G (HLA-G) to inhibit OGvHD. A major histocompatibility mismatch chronic OGvHD murine model was evaluated. 7 days after HSCT, mice were dosed subconjunctivally with scAAV8-HLA-G1/5 (1 x 109 vg/eye), topical cyclosporine (twice daily), or left untreated. Body weights and tear production (red thread test) were recorded, and eyelid, corneal opacity, and corneal fluorescein retention were scored through day 44 after HSCT. Tissues were collected for vector biodistribution, ocular histology, and immunofluorescence. Compared with untreated HSCT eyes, those dosed with scAAV8-HLA-G1/5 had significantly reduced clinical inflammatory signs of OGvHD. On histology, eyes that received scAAV8-HLA-G1/5 or cyclosporine had a significantly lower mean limbal mononuclear cell count when compared with non-treated HSCT eyes. HLA-G immunofluorescence was detected in the subconjunctiva and peripheral cornea in HSCT animals treated with scAAV8-HLA-G1/5. Vector genomes were detected in the lacrimal gland, but not in the other tested organs. These results provide evidence that subconjunctival AAV targets ocular surface and corneal disease and support that HLA-G-based gene therapy may be an effective treatment for OGvHD.

2.
J Mater Sci ; 58(7): 3360-3374, 2023.
Article in English | MEDLINE | ID: mdl-36817314

ABSTRACT

In this study, the extreme tension wave front profile (pull speed up to 1.6 km/s) in pure aluminum (density 2.7  g / cm 3 ) is analyzed using the LAMMPS molecular dynamics (MD) code and based on the tension conservation equations of mass, momentum, and energy. The simulation results agree favorably with the theoretical calculation. The profile of the extreme tension wave front is observed from the MD code simulation, and a typical shockless ramp wave front formation is identified during forced extreme tension loading. Further analysis was accomplished based on the formation of the ramp wave front, illustrating the behavior of the isentrope of aluminum under extreme tension loading.

3.
PLoS One ; 17(8): e0270972, 2022.
Article in English | MEDLINE | ID: mdl-35980983

ABSTRACT

Equine recurrent uveitis (ERU) is a spontaneous, painful, and vision threatening disease affecting up to 25% of equine populations worldwide. Current treatments of ERU are non-specific and have many side effects which limits them to short-term use. In order to develop an effective therapy for ERU, we investigated the use of adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by equine interleukin-10 (Equine-IL10). The purpose of this study was to evaluate the therapeutic efficacy of a single intravitreal (IVT) dose of AAV8-Equine-IL10 gene therapy for inhibition of experimental autoimmune uveitis (EAU) in rats. Each rat was dosed intravitreally (IVT) in both eyes with either balanced salt solution (BSS) (control; n = 4), AAV8-Equine-IL10 at a low dose (2.4x109 vg; n = 5) or high dose (2.4x1010 vg; n = 5). EAU was induced in all groups of rats 7 days after IVT injections and euthanized 21 days post-injection. Ophthalmic examination and aqueous humor (AH) cell counts were recorded with the observer blinded to the treatment groups. Histopathology and qPCR were performed on selected ocular tissues. Data presented herein demonstrate that AAV8-Equine-IL10 treated rats exhibited a significant decrease in clinical inflammatory scores and AH cell counts compared to BSS-treated EAU eyes on days 10, 12 and 14 post EAU induction at both administered vector doses. Mean cellular histologic infiltrative scores were also significantly less in AAV8-Equine-IL10 dosed rats compared to the BSS group. Intravitreal injection of AAV8-Equine-IL10 resulted in Equine-IL10 cDNA expression in the ciliary body, retina, cornea, and optic nerve in a dose-dependent manner. A single IVT injection of AAV8-Equine-IL10 appeared to be well-tolerated and inhibited EAU even at the lowest administered dose. These results demonstrate safety and efficacy of AAV8-Equine-IL10 to prevent EAU and support continued exploration of AAV gene therapy for the treatment of equine and perhaps human recurrent uveitis.


Subject(s)
Autoimmune Diseases , Uveitis , Animals , Dependovirus/genetics , Genetic Therapy , Horses/genetics , Humans , Interleukin-10/genetics , Interleukin-10/therapeutic use , Rats
4.
Int J Mol Sci ; 23(7)2022 Mar 23.
Article in English | MEDLINE | ID: mdl-35408825

ABSTRACT

The purpose of this paper is to review human leukocyte antigen G (HLA-G) in the eye, its role in immune tolerance, and the potential therapeutic use of AAV gene transfer and expression of HLA-G in various ocular tissues. Several studies are reviewed that demonstrate efficacy in animal models of disease, including intracorneal delivery of AAV-HLA-G to treat corneal inflammation and prevent corneal graft rejection, subconjunctival injection of AAV-HLA-G for ocular graft vs. host disease and potentially dry eye disease, and intravitreal injection of AAV-HLA-G to inhibit uveitis. Furthermore, due to the anti-vascular function of HLA-G, AAV-HLA-G may be an effective therapy for posterior ocular diseases, such as neovascular age-related macular degeneration, diabetic retinopathy, and choroidal neovascularization. Therefore, AAV-mediated gene transfer of HLA-G may be an effective treatment for common immune-mediated, inflammatory, and neovascular diseases of the eye.


Subject(s)
Choroidal Neovascularization , Dependovirus , Animals , Choroidal Neovascularization/genetics , Dependovirus/genetics , Genetic Therapy , Genetic Vectors/genetics , HLA-G Antigens/genetics
5.
Viruses ; 13(7)2021 06 23.
Article in English | MEDLINE | ID: mdl-34201599

ABSTRACT

Adeno-associated virus (AAV) was first characterized as small "defective" contaminant particles in a simian adenovirus preparation in 1965. Since then, a recombinant platform of AAV (rAAV) has become one of the leading candidates for gene therapy applications resulting in two FDA-approved treatments for rare monogenic diseases and many more currently in various phases of the pharmaceutical development pipeline. Herein, we summarize rAAV approaches for the treatment of diverse types of cancers and highlight the natural anti-oncogenic effects of wild-type AAV (wtAAV), including interactions with the cellular host machinery, that are of relevance to enhance current treatment strategies for cancer.


Subject(s)
Dependovirus/physiology , Genetic Therapy , Neoplasms/therapy , Cell Death , Clinical Trials as Topic , Combined Modality Therapy , Dependovirus/genetics , Drug Therapy , Genetic Vectors , Humans , Neoplasms/genetics , Neoplasms/pathology , Neoplasms/virology , Neoplastic Stem Cells/pathology , Neoplastic Stem Cells/virology , Terminal Repeat Sequences , Viral Proteins/metabolism
6.
Am J Otolaryngol ; 42(1): 102764, 2021.
Article in English | MEDLINE | ID: mdl-33096338

ABSTRACT

OBJECTIVES: Recognize the avoidable costs incurred due to overpacking of rhinoplasty instrument trays. Reduce rhinoplasty instrument trays by including only instruments used frequently. Establish methods to reduce trays prepared for other otolaryngologic procedures. METHODS: This is a prospective study. The study evaluates the specific use of instruments opened for rhinoplasty procedures at the New York Eye & Ear Infirmary of Mount Sinai. Instruments were counted in 10 rhinoplasty cases. Usage rate was calculated for each instrument. Additionally, all instruments used in at least 20% of cases were noted. This "20%" threshold was used to create new rhinoplasty tray inventories more reflective of actual instrument usage. Some instruments above the 20% threshold were included in multiples (i.e. two Adson Brown forceps vs. one curved iris scissor). RESULTS: 189 instruments were opened, and 32 instruments were used on average in each rhinoplasty. 55 instruments were used in at least 20% of cases. The 55 "high usage" instruments were used to create new, reduced rhinoplasty tray inventory lists. Based on our analysis, a new rhinoplasty tray inventory was created comprised of 68 instruments, a 64% reduction from 189. CONCLUSION: Instruments are sterilized and packed in gross excess for rhinoplasty procedures. Previously published figures estimate re-sterilization costs of $0.51 to $0.77 per instrument. Reduction in instruments opened from 189 to 68 is expected to lead to cost savings ranging from $62 to $93 per case, yielding a savings between $6200 and $9300 per 100 cases performed. LEVEL OF EVIDENCE: II-3.


Subject(s)
Rhinoplasty/instrumentation , Surgical Instruments/economics , Surgical Instruments/statistics & numerical data , Utilization Review , Cost Savings/economics , Prospective Studies , Rhinoplasty/economics , Sterilization/economics
7.
Laryngoscope ; 131(2): E420-E422, 2021 02.
Article in English | MEDLINE | ID: mdl-32767559

ABSTRACT

The prevalence of residual epiphora following successful periocular surgery for facial nerve paralysis can be as high as 30% or more. The pathophysiology of residual epiphora is complex, but identification of the etiology is paramount because the therapeutic approach varies accordingly. Treatments range from medical management of systemic disease to botulinum toxin injections for conditions that arise from aberrant reinnervation to surgical procedures that bypass the lacrimal drainage system completely. We describe a case report and review the pathophysiology and management of residual epiphora to provide a treatment algorithm for clinical use by facial plastic and oculoplastic surgeons. Laryngoscope, 131:E420-E422, 2021.


Subject(s)
Facial Paralysis/surgery , Lacrimal Apparatus Diseases/etiology , Cranial Nerve Neoplasms/surgery , Facial Nerve/surgery , Facial Nerve Diseases/surgery , Female , Humans , Lacrimal Apparatus Diseases/physiopathology , Lacrimal Apparatus Diseases/therapy , Middle Aged , Tears/physiology
8.
Pharmaceutics ; 12(8)2020 Aug 13.
Article in English | MEDLINE | ID: mdl-32823625

ABSTRACT

According to the World Health Organization, corneal diseases are the fourth leading cause of blindness worldwide accounting for 5.1% of all ocular deficiencies. Current therapies for corneal diseases, which include eye drops, oral medications, corrective surgeries, and corneal transplantation are largely inadequate, have undesirable side effects including blindness, and can require life-long applications. Adeno-associated virus (AAV) mediated gene therapy is an optimistic strategy that involves the delivery of genetic material to target human diseases through gene augmentation, gene deletion, and/or gene editing. With two therapies already approved by the United States Food and Drug Administration and 200 ongoing clinical trials, recombinant AAV (rAAV) has emerged as the in vivo viral vector-of-choice to deliver genetic material to target human diseases. Likewise, the relative ease of applications through targeted delivery and its compartmental nature makes the cornea an enticing tissue for AAV mediated gene therapy applications. This current review seeks to summarize the development of AAV gene therapy, highlight preclinical efficacy studies, and discuss potential applications and challenges of this technology for targeting corneal diseases.

9.
Hum Gene Ther ; 31(19-20): 1054-1067, 2020 10.
Article in English | MEDLINE | ID: mdl-32829671

ABSTRACT

Recombinant adeno-associated viral (rAAV) vector mobilization is a largely theoretical process in which intact AAV vectors spread or "mobilize" from transduced cells and infect additional cells within, or external of, the initial host. This process can be helper virus-independent (vector alone) or helper virus-dependent (de novo rAAV production facilitated by superinfection of both wild-type AAV [wtAAV] and Adenovirus 5 [Ad] helper virus). Herein, rAAV production and mobilization with and without wtAAV were analyzed following plasmid transfection or viral transduction utilizing well-established in vitro conditions and analytical measurements. During in vitro production, wtAAV produced the highest titer with rAAV-luc (4.1 kb), rAAV-IDUA (3.7 kb), and rAAV-Nano-dysferlin (4.9 kb) generating 2.5-, 5.9-, or 10.7-fold lower amounts, respectively. Surprisingly, cotransfection of a wtAAV and an rAAV plasmid resulted in a uniform decrease in production of wtAAV in all instances with a concomitant increase of rAAV such that wtAAV:rAAV titers were at a ratio of 1:1 for all constructs investigated. These results were shown to be independent of the rAAV transgenic sequence, size, transgene, or promoter choice and point to novel aspects of wtAAV complementation that enhance current vector production systems yet to be defined. In a mobilization assay, a sizeable amount of rAAV recovered from infected 293 cell lysate remained intact and competent for a secondary round of infection (termed Ad-independent mobilization). In rAAV-infected cells coinfected with Ad and wtAAV, rAAV particle production was increased >50-fold compared with no Ad conditions. In addition, Ad-dependent rAAV vectors mobilized and resulted in >1,000-fold transduction upon a subsequent second-round infection, highlighting the reality of these theoretical safety concerns that can be manifested under various conditions. Overall, these studies document and signify the need for mobilization-resistant vectors and the opportunity to derive better vector production systems.


Subject(s)
Adenoviridae/genetics , DNA Replication , DNA, Viral/genetics , Dependovirus/physiology , Genetic Vectors/administration & dosage , Recombination, Genetic , Virus Assembly , Genetic Vectors/genetics , HeLa Cells , Humans
10.
Mol Ther Methods Clin Dev ; 18: 24-32, 2020 Sep 11.
Article in English | MEDLINE | ID: mdl-32542182

ABSTRACT

The chronic ocular toxicity, tolerability, and inflammation following corneal intrastromal injection of saline or escalating doses of an adeno-associated virus (AAV) containing a codon-optimized α-l-iduronidase (AAV-opt-IDUA) expression cassette were evaluated in New Zealand White rabbits. Corneal opacity following corneal intrastromal injection resolved by 24 h. Mild elevation of clinical ocular inflammation was observed 24 h after injection, but it returned to baseline by day 7 and no abnormalities were noted through 6 months of observation after injection. Vector genomes and IDUA cDNA were detected in the injected corneas in a dose-dependent manner. Both the lowest administered AAV-opt-IDUA dose, shown to be effective in mucopolysaccharidosis type I (MPS I) dogs, and a 10-fold higher dose of AAV-opt-IDUA resulted in no detectable immunologic response or adverse effect in rabbits. Vector genomes outside of the eye were rarely detected following corneal intrastromal injection of AAV-opt-IDUA, and neutralizing antibodies to the AAV capsid were not present at the experimental conclusion. This study, combined with our previous studies in MPS I dogs, suggests that AAV-opt-IDUA corneal gene therapy following corneal intrastromal injection of AAV-opt-IDUA has the potential to prevent and reverse blindness in MPS I patients in a safe and effective manner.

11.
Methods Mol Biol ; 2145: 77-102, 2020.
Article in English | MEDLINE | ID: mdl-32542602

ABSTRACT

Gene delivery approaches using adeno-associated virus (AAV) vectors are currently the preferred method for human gene therapy applications and have demonstrated success in clinical trials for a diverse set of diseases including retinal blindness. To date, no clinical trials using AAV gene therapy in the anterior eye have been initiated; however, corneal gene delivery appears to be an attractive approach for treating both corneal and ocular surface diseases. Multiple preclinical studies by our lab and others have demonstrated efficient AAV vector-mediated gene delivery to the cornea for immunomodulation, anti-vascularization, and enzyme supplementation. Interestingly, the route of AAV vector administration and nuances such as administered volume influence vector tropism and transduction efficiency. In this chapter, a detailed protocol for AAV vector production and specific approaches for AAV-mediated gene transfer to the cornea via subconjunctival and intrastromal injections are described.


Subject(s)
Cornea/growth & development , Eye Diseases/genetics , Genetic Therapy/methods , Transduction, Genetic/methods , Animals , Cornea/pathology , Dependovirus/genetics , Eye Diseases/therapy , Gene Transfer Techniques , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , HEK293 Cells , Humans , Retina/growth & development , Retina/pathology , Transgenes/genetics
12.
Mol Ther ; 28(6): 1455-1463, 2020 06 03.
Article in English | MEDLINE | ID: mdl-32330426

ABSTRACT

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disease characterized by severe phenotypes, including corneal clouding. MPS I is caused by mutations in alpha-l-iduronidase (IDUA), a ubiquitous enzyme that catalyzes the hydrolysis of glycosaminoglycans. Currently, no treatment exists to address MPS I corneal clouding other than corneal transplantation, which is complicated by a high risk for rejection. Investigation of an adeno-associated virus (AAV) IDUA gene addition strategy targeting the corneal stroma addresses this deficiency. In MPS I canines with early or advanced corneal disease, a single intrastromal AAV8G9-IDUA injection was well tolerated at all administered doses. The eyes with advanced disease demonstrated resolution of corneal clouding as early as 1 week post-injection, followed by sustained corneal transparency until the experimental endpoint of 25 weeks. AAV8G9-IDUA injection in the MPS I canine eye with early corneal disease prevented the development of advanced corneal changes while restoring clarity. Biodistribution studies demonstrated vector genomes in ocular compartments other than the cornea and in some systemic organs; however, a capsid antibody response was detected in only the highest dosed subject. Collectively, the results suggest that intrastromal AAV8G9-IDUA therapy prevents and reverses visual impairment associated with MPS I corneal clouding.


Subject(s)
Corneal Diseases/etiology , Corneal Diseases/therapy , Gene Transfer Techniques , Genetic Therapy , Mucopolysaccharidosis I/complications , Mucopolysaccharidosis I/genetics , Animals , Animals, Genetically Modified , Corneal Diseases/diagnosis , Dependovirus/genetics , Disease Models, Animal , Dogs , Female , Fluorescent Antibody Technique , Gene Expression , Gene Knockdown Techniques , Genes, Reporter , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Iduronidase/genetics , Male , Transgenes , Treatment Outcome
13.
Hum Gene Ther ; 31(3-4): 151-162, 2020 02.
Article in English | MEDLINE | ID: mdl-31914802

ABSTRACT

Adeno-associated viral vectors have been successfully used in laboratory and clinical settings for efficient gene delivery. In these vectors, 96% of the adeno-associated virus (AAV) genome is replaced with a gene cassette of interest, leaving only the 145 bp inverted terminal repeat (ITR) sequences. These cis-elements, primarily from AAV serotype 2, are required for genome rescue, replication, packaging, and vector persistence. Previous work from our lab and others have demonstrated that the AAV ITR2 sequence has inherent transcriptional activity, which may confound intended transgene expression in therapeutic applications. Currently, AAV capsids are extensively study for vector contribution; however, a comprehensive analysis of ITR promoter activity of various AAV serotypes has not been described to date. Here, the transcriptional activity of AAV ITRs from different serotypes (1-4, 6, and 7) was compared in numerous cell lines and a mouse model. Under the conditions used here, all ITRs tested were capable of promoting transgene expression both in vitro and in vivo. However, we observed three classes of AAV ITR expression in vitro. Class I ITRs (AAV2 and 3) generated the highest level, whereas class II (AAV 4) had intermediate levels, and class III (AAV1 and 6) had the lowest levels. These expression levels were consistent across multiple cell lines. Only ITR7 demonstrated cell-type dependent transcriptional activity. In vivo, all classes had promoter activity. Next-generation sequencing revealed multiple transcriptional start sites that originated from the ITR sequence, with most arising from within the Rep binding element. The collective results demonstrate that the serotype ITR sequence may have multiple levels of influence on transgene expression cassettes independent of promoter selection.


Subject(s)
Dependovirus/genetics , Gene Expression , Genetic Vectors/genetics , Terminal Repeat Sequences , Transgenes , Animals , Base Sequence , Cell Line , Dependovirus/classification , Gene Expression Regulation, Viral , Gene Transfer Techniques , Genes, Reporter , Genetic Engineering , Genetic Variation , Genetic Vectors/biosynthesis , Humans , Mice , Nucleic Acid Conformation , Plasmids/genetics , Promoter Regions, Genetic , Serogroup , Transcription Initiation Site , Transcriptional Activation , Transduction, Genetic
14.
Cornea ; 39(3): 362-369, 2020 Mar.
Article in English | MEDLINE | ID: mdl-31724981

ABSTRACT

PURPOSE: Drug delivery directly to the corneal stroma currently relies on microscopic injections that demonstrate low reproducibility and clinician-dependent variability. With use of biological drugs such as adeno-associated viral (AAV) vectors, precise and consistent drug deposition is critical to reduce concerns related to off-target transduction and the host's immune response to the viral capsid and/or transgene-derived product. Therefore, a precise corneal injection (PCI) microneedle was designed to allow accurate depth-specific injections into the corneal stroma in a macroscopic setting. METHODS: High-frequency ultrasound and confocal microscopy demonstrated the consistent ability to predetermine the precise injection depth using PCI needles of varying sizes. Next, a comparison between a standard 31-G needle and PCI needles was performed in vivo using AAV vector gene delivery. RESULTS: Intrastromal corneal injections using the PCI microneedle resulted in less vector leakage at the site of injection and fewer anterior chamber penetrations compared with a standard 31-G needle. Although reporter gene expression appeared similar when the vector was administered with either needle type, a trend toward increased vector genomes was noted in the PCI-injected corneas at the experimental conclusion. As hypothesized, corneal perforation resulted in increased detection of AAV vector genomes in nontarget tissues, highlighting the importance of consistency for biological drug applications in the cornea. CONCLUSIONS: Further development of the PCI microneedle is warranted especially for AAV corneal gene therapy and offers the potential to enhance transduction while significantly reducing safety concerns and intraclinician and interclinician injection variability.


Subject(s)
Corneal Stroma/metabolism , Dependovirus/genetics , Genetic Therapy/methods , Genetic Vectors , Green Fluorescent Proteins/genetics , Needles , Animals , Gene Expression , Gene Transfer Techniques , Injections, Intraocular , Male , Microscopy, Confocal , Rabbits , Reproducibility of Results , Swine , Ultrasonography
15.
Sci Rep ; 9(1): 19864, 2019 12 27.
Article in English | MEDLINE | ID: mdl-31882729

ABSTRACT

Non-infectious uveitis (NIU) is an intractable, recurrent, and painful disease that is a common cause of vision loss. Available treatments of NIU, such as the use of topical corticosteroids, are non-specific and have serious side effects which limits them to short-term use; however, NIU requires long-term treatment to prevent vision loss. Therefore, a single dose therapeutic that mediates long-term immunosuppression with minimal side effects is desirable. In order to develop an effective long-term therapy for NIU, an adeno-associated virus (AAV) gene therapy approach was used to exploit a natural immune tolerance mechanism induced by the human leukocyte antigen G (HLA-G). To mimic the prevention of NIU, naïve Lewis rats received a single intravitreal injection of AAV particles harboring codon-optimized cDNAs encoding HLA-G1 and HLA-G5 isoforms one week prior to the induction of experimental autoimmune uveitis (EAU). AAV-mediated expression of the HLA-G-1 and -5 transgenes in the targeted ocular tissues following a single intravitreal injection of AAV-HLA-G1/5 significantly decreased clinical and histopathological inflammation scores compared to untreated EAU eyes (p < 0.04). Thus, localized ocular gene delivery of AAV-HLA-G1/5 may reduce the off-target risks and establish a long-term immunosuppressive effect that would serve as an effective and novel therapeutic strategy for NIU, with the potential for applications to additional ocular immune-mediated diseases.


Subject(s)
Dependovirus/genetics , HLA-G Antigens/metabolism , HLA-G Antigens/physiology , Uveitis/pathology , Uveitis/therapy , Animals , Antibodies, Neutralizing/metabolism , Female , Genetic Therapy , HLA-G Antigens/genetics , Intravitreal Injections , Rats , Uveitis/genetics , Uveitis/metabolism
16.
Hum Gene Ther ; 30(11): 1336-1348, 2019 11.
Article in English | MEDLINE | ID: mdl-31392914

ABSTRACT

Limbal stem cell (LSC) transplantation is a promising treatment for ocular surface diseases especially LSC deficiency. Genetic engineering represents an attractive strategy to increase the potential for success in LSC transplantations either by correcting autologous diseased LSCs or by decreasing the immunogenicity of allogeneic LSCs. Therefore, two popular viral vectors, adeno-associated viral (AAV) vector and lentiviral (LV) vector, were compared for gene delivery in human LSCs. Transduction efficiency was evaluated by flow cytometry, quantitation of viral genomes, and fluorescence microscopy after introducing eight self-complementary AAV serotypes or LV carrying a green fluorescent protein (GFP) cassette to fresh limbal epithelial cells, cultivated LSC colonies, or after corneal intrastromal injection into human explant tissue. For fresh limbal epithelial cells, AAV6 showed the highest transduction efficiency, followed by LV and AAV4 at 24 h after vector incubation, which did not directly correlate with internalized genome copy number. The colony formation efficiency, as well as colony size over time, showed no significant differences among AAV serotypes, LV, and nontreated controls. The percentage of GFP+ colonies at 14 days post-seeding was significantly higher in the LV group, which plateaued at 50% GFP+ upon serial passages. Interestingly, AAV6-treated colonies initially showed a variegated transduction phenotype with no GFP+ colonies in serial passages. Quantitative polymerase chain reaction and AAV6 capsid staining revealed that transduction was restricted to differentiated cells of LSC colonies at a post-entry step. Following central intrastromal injection of human corneas, both LV and AAV6 transduced the stroma and endothelial cells, and AAV6 also transduced cells of the epithelia. However, no transduction was observed in derived LSC colonies. The collective results demonstrate the effectiveness of LV for stable human LSC genetic engineering and an unreported phenomenon of AAV6 transduction restriction in multipotent cells derived from the human limbus.


Subject(s)
Dependovirus/genetics , Gene Transfer Techniques , Genetic Vectors/administration & dosage , Limbus Corneae/cytology , Stem Cells/metabolism , Animals , Cell Differentiation , Dependovirus/physiology , Endothelial Cells/metabolism , Green Fluorescent Proteins/metabolism , Humans , Mice , NIH 3T3 Cells , Transgenes , Virus Internalization
17.
Facial Plast Surg ; 35(1): 90-95, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30566989

ABSTRACT

Social determinants of health have been widely studied throughout medicine; however, their role relating to functional rhinoplasty has not been previously evaluated. The records of 178 patients who underwent functional rhinoplasty in a single health network from 2013 to 2016 were reviewed. The Nasal Obstruction Symptom Evaluation (NOSE) score was used to assess patient-reported symptoms, and patients with both preoperative and postoperative NOSE scores were included in this study. Basic demographics and surgical techniques were also collected. Differences between NOSE scores and surgical approaches to functional rhinoplasty on the basis of insurance type were measured. One hundred and sixteen patients were included for analysis, the mean age was 34.7 years (standard deviation [SD] = 16.2) and 57 (49.1%) were female. Twenty-one (18.1%) patients had public insurance and, of these, 18 patients had Medicaid. Patients (mean, SD) with Medicaid insurance (56.39, 15.6) had a slightly greater improvement in NOSE scores compared with patients with non-Medicaid insurance (47.90, 25.6) (p = 0.067). There was no statistically significant difference in preoperative NOSE scores or postoperative improvement in NOSE scores between patients with different health insurance. Furthermore, there was no statistically significant difference in surgical approaches. The majority of patients receiving functional rhinoplasty had private insurance. Medicaid patients trended toward a greater NOSE score improvement after functional rhinoplasty, but also had a closer association with a history of nasal trauma and prior surgery. Future study is needed to better understand the association between socioeconomic status and disparities in care. Understanding how social determinants of health affect patients may reveal potential inherent biases, improve delivery of care, and translate to better patient outcomes.


Subject(s)
Insurance, Health/statistics & numerical data , Medicaid/statistics & numerical data , Nasal Obstruction/surgery , Rhinoplasty , Adolescent , Adult , Female , Health Status Disparities , Healthcare Disparities , Humans , Male , Middle Aged , Postoperative Period , Preoperative Period , Rhinoplasty/methods , Severity of Illness Index , United States , Young Adult
18.
Gene Ther ; 25(6): 402-414, 2018 09.
Article in English | MEDLINE | ID: mdl-30072815

ABSTRACT

AAV gene therapy approaches in the posterior eye resulted in the first FDA-approved gene therapy-based drug. However, application of AAV vectorology to the anterior eye has yet to enter even a Phase I trial. Furthermore, the simple and safe subconjunctival injection has been relatively unexplored in regard to AAV vector transduction. To determine the utility of this route for the treatment of various ocular disorders, a survey of gene delivery via natural AAV serotypes was performed and correlated to reported cellular attachment factors. AAV serotypes packaged with a self-complementary reporter were administered via subconjunctival injection to WT mice. Subconjunctival injection of AAV vectors was without incidence; however, vector shedding in tears was noted weeks following administration. AAV transduction was serotype dependent in anterior segment tissues including the eye lid, conjunctiva, and cornea, as well as the periocular tissues including muscle. Transgene product in the cornea was highest for AAV6 and AAV8, however, their corneal restriction was remarkably different; AAV6 appeared restricted to the endothelium layer while AAV8 efficiently transduced the stromal layer. Reported AAV cellular receptors were not well correlated to vector transduction; although, in some cases they were conserved among mouse and human ocular tissues. Subconjunctival administration of particular AAV serotypes may be a simple and safe targeted gene delivery route for ocular surface, muscular, corneal, and optic nerve diseases.


Subject(s)
Dependovirus/genetics , Eye Diseases/therapy , Gene Transfer Techniques , Genetic Vectors/genetics , Animals , Conjunctiva/pathology , Cornea/metabolism , Cornea/pathology , Cornea/virology , Eye Diseases/genetics , Eye Diseases/pathology , Genetic Therapy , Genetic Vectors/immunology , Green Fluorescent Proteins/genetics , Humans , Mice , Serogroup , Surveys and Questionnaires , Transduction, Genetic
19.
JCI Insight ; 3(12)2018 06 21.
Article in English | MEDLINE | ID: mdl-29925692

ABSTRACT

Data from clinical trials for hemophilia B using adeno-associated virus (AAV) vectors have demonstrated decreased transgenic coagulation factor IX (hFIX) expression 6-10 weeks after administration of a high vector dose. While it is likely that capsid-specific cytotoxic T lymphocytes eliminate vector-transduced hepatocytes, thereby resulting in decreased hFIX, this observation is not intuitively consistent with restored hFIX levels following prednisone application. Although the innate immune response is immediately activated following AAV vector infection via TLR pathways, no studies exist regarding the role of the innate immune response at later time points after AAV vector transduction. Herein, activation of the innate immune response in cell lines, primary human hepatocytes, and hepatocytes in a human chimeric mouse model was observed at later time points following AAV vector transduction. Mechanistic analysis demonstrated that the double-stranded RNA (dsRNA) sensor MDA5 was necessary for innate immune response activation and that transient knockdown of MDA5, or MAVS, decreased IFN-ß expression while increasing transgene production in AAV-transduced cells. These results both highlight the role of the dsRNA-triggered innate immune response in therapeutic transgene expression at later time points following AAV transduction and facilitate the execution of effective strategies to block the dsRNA innate immune response in future clinical trials.


Subject(s)
Dependovirus/genetics , Genetic Vectors/immunology , Immunity, Innate/immunology , Parvoviridae Infections/immunology , RNA, Double-Stranded/immunology , Transduction, Genetic , Animals , Capsid , Cell Line , Factor IX/genetics , Factor IX/metabolism , Gene Knockdown Techniques , Genetic Therapy , Genetic Vectors/genetics , HEK293 Cells , HeLa Cells , Hep G2 Cells , Hepatocytes/immunology , Humans , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/immunology , Interferon-beta/metabolism , Liver/metabolism , Mice , Models, Animal , RNA, Double-Stranded/genetics , Transgenes/genetics , Transplantation, Heterologous
20.
Hum Mol Genet ; 27(4): 601-613, 2018 02 15.
Article in English | MEDLINE | ID: mdl-29272432

ABSTRACT

The clinical trial using adeno-associated virus (AAV) vector delivery of mini-dystrophin in patients with Duchenne Muscular Dystrophy (DMD) demonstrated a cytotoxic lymphocyte (CTL) response targeting the transgene product. These mini-dystrophin-specific T-cells have the potential to clear all transduced muscle, presenting the general gene therapy concern of overcoming the CTL response to foreign proteins that provide therapeutic benefit. In this study, we exploited a natural immunosuppression strategy employed by some viruses that results in CTL evasion only in transduced cells. After transfection of the plasmids encoding viral peptides and ovalbumin, which includes the immune-domain epitope SIINFEKL, several viral small peptides (ICP47 and US6) inhibited the SIINFEKL peptide presentation. A single AAV vector genome that consisted of either transgene AAT fused with SIINFEKL epitope and, separately, ICP47 expressed from different promoters or a single fusion protein with ICP47 linked by a furin cleavage peptide (AATOVA-ICP47) decreased antigen presentation. Compared with AAV/AATOVA in which decreased AAT expression was observed at late time points, persistent transgene expression was obtained after systemic administration of AAV/AATOVA-ICP47 vectors in mice. We extended this strategy to DMD gene therapy. After administration of AAV vector encoding human mini-dystrophin fusion protein with ICP47 into mdx mice, a lower mini-dystrophin-specific CTL response was induced. Importantly, the ICP47 fusion to mini-dystrophin inhibited CTLs mediated cytotoxicity. Although demonstrated herein using AAT and mini-dystrophin transgenes in an AAV context, the collective results have implications for all gene therapy applications resulting in foreign peptides by immune suppression in only genetically modified cells.


Subject(s)
Antigen Presentation/immunology , Dependovirus/genetics , Dependovirus/immunology , Animals , Female , Genetic Therapy/methods , Male , Mice , Muscle, Skeletal/metabolism , Muscular Dystrophy, Duchenne , Peptides/immunology , Spleen/metabolism , T-Lymphocytes/metabolism , Viral Proteins/immunology , Viral Proteins/metabolism
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