Sensitive quantification of m.3243A>G mutational proportion in non-retinal tissues and its relationship with visual symptoms

Mullin NK, Anfinson KR, Riker MJ, Wieland KL, Tatro NJ, Scheetz TE, Mullins RF, Stone EM, Tucker BA.


The m.3243A>G mutation in the mitochondrial genome commonly causes retinal degeneration in patients with maternally inherited diabetes and deafness (MIDD) and mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Like other mitochondrial mutations, m.3243A>G is inherited from the mother with a variable proportion of wild type and mutant mitochondrial genomes in different cells. The mechanism by which the m.3243A>G variant in each tissue relates to the manifestation of disease phenotype is not fully understood. Using a digital PCR assay we found that the % m.3243G in skin derived dermal fibroblasts was positively correlated with that of blood from the same individual. The % m.3243G detected in fibroblast cultures remained constant over multiple passages and was negatively correlated with mtDNA copy number. Although the % m.3243G present in blood was not correlated with severity of vision loss, as quantified by Goldmann visual field, a significant negative correlation between % m.3243G and the age of onset of visual symptoms was detected. Together, these results indicate that precise measurement of % m.3243G in clinically accessible tissues such as skin and blood may yield information relevant to the management of retinal m.3243A>G associated disease.

PMID: 34590675

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

Tucker BA, Burnight ER, Cranston CM, Ulferts MJ, Luse MA, Westfall T, Scott CA, Marsden A, Gibson-Corley K, Wiley LA, Han IC, Slusarski DC, Mullins RF, Stone EM.


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


Microfluidic processing of stem cells for autologous cell replacement

Stone NE, Voight AP, Mullins RF, Sulchek T, Tucker BA.


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

Patient derived stem cells for discovery and validation of novel pathogenic variants in inherited retinal disease

Mullin NK, Voight AP, Cooke JA, Bohrer LR, Burnight ER, Stone EM, Mullins RF, Tucker BA.


Our understanding of inherited retinal disease has benefited immensely from molecular genetic analysis over the past several decades. New technologies that allow for increasingly detailed examination of a patient's DNA have expanded the catalog of genes and specific variants that cause retinal disease. In turn, the identification of pathogenic variants has allowed the development of gene therapies and low-cost, clinically focused genetic testing. Despite this progress, a relatively large fraction (at least 20%) of patients with clinical features suggestive of an inherited retinal disease still do not have a molecular diagnosis today. Variants that are not obviously disruptive to the codon sequence of exons can be difficult to distinguish from the background of benign human genetic variations. Some of these variants exert their pathogenic effect not by altering the primary amino acid sequence, but by modulating gene expression, isoform splicing, or other transcript-level mechanisms. While not discoverable by DNA sequencing methods alone, these variants are excellent targets for studies of the retinal transcriptome. In this review, we present an overview of the current state of pathogenic variant discovery in retinal disease and identify some of the remaining barriers. We also explore the utility of new technologies, specifically patient-derived induced pluripotent stem cell (iPSC)-based modeling, in further expanding the catalog of disease-causing variants using transcriptome-focused methods. Finally, we outline bioinformatic analysis techniques that will allow this new method of variant discovery in retinal disease. As the knowledge gleaned from previous technologies is informing targets for therapies today, we believe that integrating new technologies, such as iPSC-based modeling, into the molecular diagnosis pipeline will enable a new wave of variant discovery and expanded treatment of inherited retinal disease.

PMID: 33130253

Stepwise differentiation and functional characterization of human induced pluripotent stem cell-derived choroidal endothelial cells

Mulfaul K, Giacalone JC, Voigt AP, Riker MJ, Ochoa D, Han IC, Stone EM, Mullins RF, Tucker BA.


Background: Endothelial cells (ECs) are essential regulators of the vasculature, lining arteries, veins, and capillary beds. While all ECs share a number of structural and molecular features, heterogeneity exists depending on their resident tissue. ECs lining the choriocapillaris in the human eye are lost early in the pathogenesis of age-related macular degeneration (AMD), a common and devastating form of vision loss. In order to study the mechanisms leading to choroidal endothelial cell (CEC) loss and to develop reagents for repairing the choroid, a reproducible in vitro model, which closely mimic CECs, is needed. While a number of protocols have been published to direct induced pluripotent stem cells (iPSCs) into ECs, the goal of this study was to develop methods to differentiate iPSCs into ECs resembling those found in the human choriocapillaris specifically.

Methods: We transduced human iPSCs with a CDH5p-GFP-ZEO lentiviral vector and selected for transduced iPSCs using blasticidin. We generated embryoid bodies (EBs) from expanded iPSC colonies and transitioned from mTESR™1 to EC media. One day post-EB formation, we induced mesoderm fate commitment via addition of BMP-4, activin A, and FGF-2. On day 5, EBs were adhered to Matrigel-coated plates in EC media containing vascular endothelial cell growth factor (VEGF) and connective tissue growth factor (CTGF) to promote CEC differentiation. On day 14, we selected for CECs using either zeocin resistance or anti-CD31 MACS beads. We expanded CECs post-selection and performed immunocytochemical analysis of CD31, carbonic anhydrase IV (CA4), and RGCC; tube formation assays; and transmission electron microscopy to access vascular function.

Results: We report a detailed protocol whereby we direct iPSC differentiation toward mesoderm and utilize CTGF to specify CECs. The CDH5p-GFP-ZEO lentiviral vector facilitated the selection of iPSC-derived ECs that label with antibodies directed against CD31, CA4, and RGCC; form vascular tubes in vitro; and migrate into empty choroidal vessels. CECs selected using either antibiotic selection or CD31 MACS beads showed similar characteristics, thereby making this protocol easily reproducible with or without lentiviral vectors.

Conclusion: ECs generated following this protocol exhibit functional and biochemical characteristics of CECs. This protocol will be useful for developing in vitro models toward understanding the mechanisms of CEC loss early in AMD.

PMID: 32967716

Autologous cell replacement: a noninvasive AI approach to clinical release testing.

Tucker BAMullins RFStone EM.


The advent of human induced pluripotent stem cells (iPSCs) provided a means for avoiding ethical concerns associated with the use of cells isolated from human embryos. The number of labs now using iPSCs to generate photoreceptor, retinal pigmented epithelial (RPE), and-more recently-choroidal endothelial cells has grown exponentially. However, for autologous cell replacement to be effective, manufacturing strategies will need to change. Many tasks carried out by hand will need simplifying and automating. In this issue of the JCI, Schaub and colleagues combined quantitative bright-field microscopy and artificial intelligence (deep neural networks and traditional machine learning) to noninvasively monitor iPSC-derived graft maturation, predict donor cell identity, and evaluate graft function prior to transplantation. This approach allowed the authors to preemptively identify and remove abnormal grafts. Notably, the method is (a) transferable, (b) cost and time effective, (c) high throughput, and (d) useful for primary product validation.

PMID: 31961338

Development of a molecularly stable gene therapy vector for the treatment of RPGR- associated X-linked retinitis pigmentosa. 

Giacalone, JC., Andorf, JL., Zhang, Q., Burnight, ER., Ochoa, D., Reutzel, AJ., Collins, MM., Sheffield, VC., Mullins, RF., Han, IC., Stone, EM., Tucker, BA.


In a screen of 1,000 consecutively ascertained families, we recently found that mutations in the gene RPGR are the third most common cause of all inherited retinal disease. As the two most frequent disease-causing genes, ABCA4 and USH2A, are far too large to fit into clinically relevant adeno-associated virus (AAV) vectors, RPGR is an obvious early target for AAV-based ocular gene therapy. In generating plasmids for this application, we discovered that those containing wild-type RPGR sequence, which includes the highly repetitive low complexity region ORF15, were extremely unstable (i.e., they showed consistent accumulation of genomic changes during plasmid propagation). To develop a stable RPGR gene transfer vector, we used a bioinformatics approach to identify predicted regions of genomic instability within ORF15 (i.e., potential non-B DNA conformations). Synonymous substitutions were made in these regions to reduce the repetitiveness and increase the molecular stability while leaving the encoded amino acid sequence unchanged. The resulting construct was subsequently packaged into AAV serotype 5, and the ability to drive transcript expression and functional protein production was demonstrated via subretinal injection in rat and pull-down assays, respectively. By making synonymous substitutions within the repetitive region of RPGR, we were able to stabilize the plasmid and subsequently generate a clinical-grade gene transfer vector (IA-RPGR). Following subretinal injection in rat, we demonstrated that the augmented transcript was expressed at levels similar to wild-type constructs. By performing in vitro pull-down experiments, we were able to show that IA-RPGR protein product retained normal protein binding properties (i.e., analysis revealed normal binding to PDE6D, INPP5E, and RPGRIP1L). In summary, we have generated a stable RPGR gene transfer vector capable of producing functional RPGR protein, which will facilitate safety and toxicity studies required for progression to an Investigational New Drug application.


Two-photon polymerized poly(caprolactone) retinal cell delivery scaffolds and thier systemic and retinal biocompatibility. 

Thompson, JR., Worthington, KS., Green. BJ., Mullin, NK., Jiao, C., Kaalberg, EE., Wiley, LA., Han, Ic., Russell, SR., Sohn, EH., Guymon, CA., Mullins, RF., Stone, EM., Tucker, BA.


Cell replacement therapies are often enhanced by utilizing polymer scaffolds to improve retention or direct cell orientation and migration. Obstacles to refinement of such polymer scaffolds often include challenges in controlling the microstructure of biocompatible molecules in three dimensions at cellular scales. Two-photon polymerization of acrylated poly(caprolactone) (PCL) could offer a means of achieving precise microstructural control of a material in a biocompatible platform. In this work, we studied the effect of various formulation and two-photon polymerization parameters on minimum laser power needed to achieve polymerization, resolution, and fidelity to a target 3D model designed to be used for retinal cell replacement. Overall, we found that increasing the concentration of crosslink-able groups decreased polymerization threshold and the size of resolvable features while increasing fidelity of the scaffold to the 3D model. In general, this improvement was achieved by increasing the number of acrylate groups per prepolymer molecule, increasing the acrylated PCL concentration, or decreasing its molecular weight. Resulting two-photon polymerized PCL scaffolds successfully supported human iPSC derived retinal progenitor cells in vitro. Sub-retinal implantation of cell free scaffolds in a porcine model of retinitis pigmentosa did not cause inflammation, infection or local or systemic toxicity after one month. In addition, comprehensive ISO 10993 testing of photopolymerized scaffolds revealed a favorable biocompatibility profile. These results represent an important step towards understanding how two-photon polymerization can be applied to a wide range of biologically compatible chemistries for various biomedical applications. STATEMENT OF SIGNIFICANCE: Inherited retinal degenerative blindness results from the death of light sensing photoreceptor cells. To restore high-acuity vision a photoreceptor cell replacement strategy will likely be necessary. Unfortunately, single cell injection typically results in poor cell survival and integration post-transplantation. Polymeric biomaterial cell delivery scaffolds can be used to promote donor cell viability, control cellular polarity and increase packing density. A challenge faced in this endeavor has been developing methods suitable for generating scaffolds that can be used to deliver stem cell derived photoreceptors in an ordered columnar orientation (i.e., similar to that of the native retina). In this study we combined the biomaterial poly(caprolactone) with two-photon lithography to generate a biocompatible, clinically relevant scaffold suitable for retina cell delivery.

PMID: 31055121

Correction of NR2E3 Associated Enhanced S-cone Syndrome Patient-specific iPSCs using CRISPR-Cas9.

Bohrer, LR., Wiley, LA., Burnight, ER., Cooke, JA., Giacalone, JC., Anfinson, KR., Andorf, L., Mullins, RF., Stone, EM., Tucker, BA.


Enhanced S-cone syndrome (ESCS) is caused by recessive mutations in the photoreceptor cell transcription factor NR2E3. Loss of NR2E3 is characterized by repression of rod photoreceptor cell gene expression, over-expansion of the S-cone photoreceptor cell population, and varying degrees of M- and L-cone photoreceptor cell development. In this study, we developed a CRISPR-based homology-directed repair strategy and corrected two different disease-causing NR2E3 mutations in patient-derived induced pluripotent stem cells (iPSCs) generated from two affected individuals. In addition, one patient's iPSCs were differentiated into retinal cells and NR2E3 transcription was evaluated in CRISPR corrected and uncorrected clones. The patient's c.119-2A>C mutation caused the inclusion of a portion of intron 1, the creation of a frame shift, and generation of a premature stop codon. In summary, we used a single set of CRISPR reagents to correct different mutations in iPSCs generated from two individuals with ESCS. In doing so we demonstrate the advantage of using retinal cells derived from affected patients over artificial in vitro model systems when attempting to demonstrate pathophysiologic mechanisms of specific mutations.

PMID: 30959774

CRISPR-Cas9 genome engineering: Treating inherited retinal degeneration.

Burnight, ER.Giacalone, JC.Cooke, JA.Thompson, JR.Bohrer, LR.Chirco, KR.Drack, AV.Fingert, JH.Worthington, KS.Wiley, LA.Mullins, RF.Stone, EM.Tucker, BA.


Gene correction is a valuable strategy for treating inherited retinal degenerative diseases, a major cause of irreversible blindness worldwide. Single gene defects cause the majority of these retinal dystrophies. Gene augmentation holds great promise if delivered early in the course of the disease, however, many patients carry mutations in genes too large to be packaged into adeno-associated viral vectors and some, when overexpressed via heterologous promoters, induce retinal toxicity. In addition to the aforementioned challenges, some patients have sustained significant photoreceptor cell loss at the time of diagnosis, rendering gene replacement therapy insufficient to treat the disease. These patients will require cell replacement to restore useful vision. Fortunately, the advent of induced pluripotent stem cell and CRISPR-Cas9 gene editing technologies affords researchers and clinicians a powerful means by which to develop strategies to treat patients with inherited retinal dystrophies. In this review we will discuss the current developments in CRISPR-Cas9 gene editing in vivo in animal models and in vitro in patient-derived cells to study and treat inherited retinal degenerative diseases.

PMID: 29578069

CRISPR-Cas9-Based Genome Editing of Human Induced Pluripotent Stem Cells.

Giacalone, JC.Sharma, TP.Burnight, ER.Fingert, JF.Mullins, RF.Stone, EM.Tucker, BA.


Human induced pluripotent stem cells (hiPSCs) are the ideal cell source for autologous cell replacement. However, for patients with Mendelian diseases, genetic correction of the original disease-causing mutation is likely required prior to cellular differentiation and transplantation. The emergence of the CRISPR-Cas9 system has revolutionized the field of genome editing. By introducing inexpensive reagents that are relatively straightforward to design and validate, it is now possible to correct genetic variants or insert desired sequences at any location within the genome. CRISPR-based genome editing of patient-specific iPSCs shows great promise for future autologous cell replacement therapies. One caveat, however, is that hiPSCs are notoriously difficult to transfect, and optimized experimental design considerations are often necessary. This unit describes design strategies and methods for efficient CRISPR-based genome editing of patient- specific iPSCs. Additionally, it details a flexible approach that utilizes positive selection to generate clones with a desired genomic modification, Cre-lox recombination to remove the integrated selection cassette, and negative selection to eliminate residual hiPSCs with intact selection cassettes.

PMID: 29512106

The above are recent publications from the Tucker Stem Cell Laboratory. Please follow the article link to view the Pubmed entry and continue on to the full article text. For more publications, please visit PubMed.