Mullins Lab Publications

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Development and biological characterization of a clinical gene transfer vector for the treatment of MAK-associated retinitis pigmentosa

Tue, 2021-09-14 05:00

Gene Ther. 2021 Sep 14. doi: 10.1038/s41434-021-00291-5. Online ahead of print.

ABSTRACT

By combining next generation whole exome sequencing and induced pluripotent stem cell (iPSC) technology we found that an Alu repeat inserted in exon 9 of the MAK gene results in a loss of normal MAK transcript and development of human autosomal recessive retinitis pigmentosa (RP). Although a relatively rare cause of disease in the general population, the MAK variant is enriched in individuals of Jewish ancestry. In this population, 1 in 55 individuals are carriers and one third of all cases of recessive RP is caused by this gene. The purpose of this study was to determine if a viral gene augmentation strategy could be used to safely restore functional MAK protein as a step toward a treatment for early stage MAK-associated RP. Patient iPSC-derived photoreceptor precursor cells were generated and transduced with viral vectors containing the MAK transcript. One week after transduction, transcript and protein could be detected via rt-PCR and western blotting respectively. Using patient-derived fibroblast cells and mak knockdown zebra fish we demonstrate that over-expression of the retinal MAK transgene restored the cells ability to regulate primary cilia length. In addition, the visual defect in mak knockdown zebrafish was mitigated via treatment with the retinal MAK transgene. There was no evidence of local or systemic toxicity at 1-month or 3-months following subretinal delivery of clinical grade vector into wild type rats. The findings reported here will help pave the way for initiation of a phase 1 clinical trial for the treatment of patients with MAK-associated RP.

PMID:34518651 | DOI:10.1038/s41434-021-00291-5

Intrafamilial variability of ocular manifestations of von Hippel-Lindau disease

Fri, 2021-08-20 05:00

Ophthalmol Retina. 2021 Aug 17:S2468-6530(21)00255-4. doi: 10.1016/j.oret.2021.08.005. Online ahead of print.

ABSTRACT

In this retrospective cohort study, we describe intrafamilial phenotypic variability of retinal hemangioblastoma (RH) in families with von Hippel-Lindau (VHL) disease. Patients with molecularly-confirmed VHL evaluated at our institution were identified and records reviewed. For individuals with sufficient follow up and imaging (n=27), number and location of RHs at the initial and most recent follow up visits were recorded along with treatment method and systemic manifestations. A strategy for zonal classification of RH location was used. Intrafamilial phenotypic variation was identified in 3 families. Intrafamilial phenotypic variability of RH exists between family members with VHL with the same genetic mutation.

PMID:34416425 | DOI:10.1016/j.oret.2021.08.005

An Unusual Presentation of CLN3-Associated Batten Disease With Classic Histopathologic and Ultrastructural Findings

Tue, 2021-07-27 05:00

J Neuropathol Exp Neurol. 2021 Jul 27:nlab064. doi: 10.1093/jnen/nlab064. Online ahead of print.

NO ABSTRACT

PMID:34313756 | DOI:10.1093/jnen/nlab064

Exome-based investigation of the genetic basis of human pigmentary glaucoma

Sun, 2021-06-27 05:00

BMC Genomics. 2021 Jun 26;22(1):477. doi: 10.1186/s12864-021-07782-0.

ABSTRACT

BACKGROUND: Glaucoma is a leading cause of visual disability and blindness. Release of iris pigment within the eye, pigment dispersion syndrome (PDS), can lead to one type of glaucoma known as pigmentary glaucoma. PDS has a genetic component, however, the genes involved with this condition are largely unknown. We sought to discover genes that cause PDS by testing cohorts of patients and controls for mutations using a tiered analysis of exome data.

RESULTS: Our primary analysis evaluated melanosome-related genes that cause dispersion of iris pigment in mice (TYRP1, GPNMB, LYST, DCT, and MITF). We identified rare mutations, but they were not statistically enriched in PDS patients. Our secondary analyses examined PMEL (previously linked with PDS), MRAP, and 19 other genes. Four MRAP mutations were identified in PDS cases but not in controls (p = 0.016). Immunohistochemical analysis of human donor eyes revealed abundant MRAP protein in the iris, the source of pigment in PDS. However, analysis of MRAP in additional cohorts (415 cases and 1645 controls) did not support an association with PDS. We also did not confirm a link between PMEL and PDS in our cohorts due to lack of reported mutations and similar frequency of the variants in PDS patients as in control subjects.

CONCLUSIONS: We did not detect a statistical enrichment of mutations in melanosome-related genes in human PDS patients and we found conflicting data about the likely pathogenicity of MRAP mutations. PDS may have a complex genetic basis that is not easily unraveled with exome analyses.

PMID:34174832 | PMC:PMC8235805 | DOI:10.1186/s12864-021-07782-0

Automated segmentation of choroidal layers from 3-dimensional macular optical coherence tomography scans

Tue, 2021-06-22 05:00

J Neurosci Methods. 2021 Jun 19;360:109267. doi: 10.1016/j.jneumeth.2021.109267. Online ahead of print.

ABSTRACT

BACKGROUND: Changes in choroidal thickness are associated with various ocular diseases, and the choroid can be imaged using spectral-domain optical coherence tomography (SD-OCT) and enhanced depth imaging OCT (EDI-OCT).

NEW METHOD: Eighty macular SD-OCT volumes from 80 patients were obtained using the Zeiss Cirrus machine. Eleven additional control subjects had two Cirrus scans done in one visit along with enhanced depth imaging (EDI-OCT) using the Heidelberg Spectralis machine. To automatically segment choroidal layers from the OCT volumes, our graph-theoretic approach was utilized. The segmentation results were compared with reference standards from two independent graders, and the accuracy of automated segmentation was calculated using unsigned/signed border positioning/thickness errors and Dice similarity coefficient (DSC). The repeatability and reproducibility of our choroidal thicknesses were determined by intraclass correlation coefficient (ICC), coefficient of variation (CV), and repeatability coefficient (RC).

RESULTS: The mean unsigned/signed border positioning errors for the choroidal inner and outer surfaces are 3.39 ± 1.26 µm (mean ± standard deviation)/- 1.52 ± 1.63 µm and 16.09 ± 6.21 µm/4.73 ± 9.53 µm, respectively. The mean unsigned/signed choroidal thickness errors are 16.54 ± 6.47 µm/6.25 ± 9.91 µm, and the mean DSC is 0.949 ± 0.025. The ICC (95% confidence interval), CV, RC values are 0.991 (0.977-0.997), 2.48%, 14.25 µm for the repeatability and 0.991 (0.977-0.997), 2.49%, 14.30 µm for the reproducibility studies, respectively.

COMPARISON WITH EXISTING METHOD(S): The proposed method outperformed our previous method using choroidal vessel segmentation and inter-grader variability.

CONCLUSIONS: This automated segmentation method can reliably measure choroidal thickness using different OCT platforms.

PMID:34157370 | DOI:10.1016/j.jneumeth.2021.109267

Microfluidic processing of stem cells for autologous cell replacement

Tue, 2021-06-22 05:00

Stem Cells Transl Med. 2021 Jun 22. doi: 10.1002/sctm.21-0080. Online ahead of print.

ABSTRACT

Autologous photoreceptor cell replacement is one of the most promising approaches currently under development for the treatment of inherited retinal degenerative blindness. Unlike endogenous stem cell populations, induced pluripotent stem cells (iPSCs) can be differentiated into both rod and cone photoreceptors in high numbers, making them ideal for this application. That said, in addition to photoreceptor cells, state of the art retinal differentiation protocols give rise to all of the different cell types of the normal retina, the majority of which are not required and may in fact hinder successful photoreceptor cell replacement. As such, following differentiation photoreceptor cell enrichment will likely be required. In addition, to prevent the newly generated photoreceptor cells from suffering the same fate as the patient's original cells, correction of the patient's disease-causing genetic mutations will be necessary. In this review we discuss literature pertaining to the use of different cell sorting and transfection approaches with a focus on the development and use of novel next generation microfluidic devices. We will discuss how gold standard strategies have been used, the advantages and disadvantages of each, and how novel microfluidic platforms can be incorporated into the clinical manufacturing pipeline to reduce the complexity, cost and regulatory burden associated with clinical grade production of photoreceptor cells for autologous cell replacement.

PMID:34156760 | DOI:10.1002/sctm.21-0080