Development of a retinal-like cell model of cone dystrophy associated with mutation in the KCNV2 gene / Разработка сетчаткоподобной клеточной модели колбочковой дистрофии ассоциированной с мутациями в гене KCNV2 тема диссертации и автореферата по ВАК РФ 00.00.00, кандидат наук Алсаллум Алмакдад

• Алсаллум Алмакдад
• кандидат наук
• 2024

Оглавление диссертации кандидат наук Алсаллум Алмакдад

Table of Contents

INTRODUCTION

Relevance of the research

Purpose of the work and tasks

Scientific novelty

Theoretical and practical significance

Methodology and research methods

Statements to be defended

Author's contribution

Structure and scope of the dissertation

Approbation of the work

CHAPTER 1. LITERATURE REVIEW

1.1 Cone Dystrophy with Supernormal Rod Response (CDSRR)

1.1.1 Clinical manifestations of Cone Dystrophy with Supernormal Rod Response (CDSRR)

1.1.2 Molecular genetics of CDSRR

1.2 KCNV2 gene and pathophysiology of CDSRR

1.2.1 KCNV2 gene structure

1.2.2 KCNV2 gene expression and regulation

1.2.3 Retinal function in CDSRR

1.2.4 Pathophysiology of CDSRR

1.3 Treatment options for CDSRR and directions for future therapy

1.3.1 Adeno-associated virus (AAV)

1.3.2 AAV-mediated gene therapy for inherited retinal diseases (IRDs)

1.3.3 AAV tropism and efficacy in the retina

1.4 Inherited retinal diseases models for AAV-mediated gene therapy studies

1.4.1 Mouse models in inherited retinal diseases

1.4.1.1 Comparative analysis of mouse models for studying IRDs

1.4.1.2 Investigating KCNV2 gene function through Kv8.2 knockout mouse models • • •

1.4.1.3 Therapeutic approaches for retinal degenerative diseases in mouse models

1.4.1.4 AAV vector delivery routes for retinal gene therapy in mouse models

1.4.2 Primary human retina models as a platform for IRDs research

1.4.2.1 Human retinal architecture

1.4.2.2 Human explant cultures in retinal disease research

1.4.2.3 Human retinal explants for AAV vector evaluation in IRD studies

1.4.3 Human retinal organoid models as a platform for IRDs research

1.4.3.1 Technology and culture of human induced pluripotent stem cells

1.4.3.2 Reprogramming somatic cells into iPSCs

1.4.3.3 Development of retinal organoids from stem cells

1.4.3.4 Retinogenesis and development of cell types within retina and retinal organoids50

1.4.3.5 Development of retinal organoids approaches

1.4.3.6 Retinal organoids in the study and treatment of genetic retinal diseases

1.4.3.7 Retinal organoids as a platform for AAV-based gene therapy of inherited retinal diseases

CHAPTER 2. MATERIALS AND METHODS

2.1 Materials and reagents used in the work

2.1.1 Reagents

2.1.2 Buffer solutions

2.1.3 Instruments for mouse and human retina isolation

2.2 Clinical assessment of patients with CDSRR

2.3 Electrophysiological assessment of patients with CDSRR

2.4 Molecular Genetics of patients with CDSRR

2.5 Human retinal explants collection and culture

2.6 Transduction of human retinal explants with AAV vectors

2.7 Subretinal injection procedure on mouse models

2.8 Isolation of mouse retinas

2.9 Assessment of time points after human retina transduction with AAV using qPCR

2.10 Immunohistochemical staining of samples obtained from human and mouse retinas

2.11 Flow cytometry of transduced human retinal explants with AAV

2.12 Generation and characterization of iPSC lines

2.12.1 Isolation of peripheral blood mononuclear cells (PBMCs) from patients with CDSRR

2.13.2 Reprogramming PBMCs to iPSCs

2.12.3 Immunocytochemistry staining for characterization of iPSC lines

2.12.4 Flow Cytometry for iPSC Pluripotency Markers

2.12.5 Three-germ layer differentiation and immunocytochemistry staining of EBs

2.12.6 Karyotype analysis for iPSC lines

2.12.7 Sanger sequencing for detection the mutations in iPSC lines

2.13 Differentiation of retinal organoids from ESC/iPSC lines

2.14 Whole mount staining of retinal organoids

2.15 Dissociation and flow cytometry for retinal organoids

2.16 qPCR for retinal organoids

2.17 RPE culturing and staining

2.18 Patch clamp recording for photoreceptor-like cells in retinal organoids

2.19 Transduction of retinal organoids using AAV vectors

2.20 AAV production and quantification

2.21 Statistical analysis

CHAPTER 3. RESULTS

3.1 Clinical and subject findings in two patients with mutations of the KCNV2 gene

3.2 Molecular genetics in two patients with mutations of the KCNV2 gene

3.3 Testing and biodistribution of AAV vectors on human retinal explants

3.4 Testing and biodistribution of AAV vectors on mice retina after subretinal injection delivery

3.5 Generation of two human induced pluripotent stem cell lines from first CDSRR patient and his mother caused by KCNV2 mutation

3.6 Generation of a human induced pluripotent stem cell line the second patient carrying a compound heterozygous mutation in the KCNV2 gene

3.7 Differentiation of human embryonic pluripotent stem cells (EPSCs) into 3D retinal organoids

3.8 Morphological characterization of hESC-derived retinal organoids

3.9 Morphological characterization of iPSC-derived retinal organoids

3.10 Functional characterization of hESC and iPSC-derived retinal organoids

3.11 Testing and biodistribution of AAV vectors on hESC and ABi001-A-derived retinal

organoids

3.12 Treatment of ABi002-A-derived retinal organoids using AAV vectors

CHAPTER 4. DISCUSSION

CONCLUSIONS

MAIN RESULTS AND OUTLOOK

LIST OF ABBREVIATIONS

REFERENCES

Введение диссертации (часть автореферата) на тему «Development of a retinal-like cell model of cone dystrophy associated with mutation in the KCNV2 gene / Разработка сетчаткоподобной клеточной модели колбочковой дистрофии ассоциированной с мутациями в гене KCNV2»

INTRODUCTION

Relevance of the research

Cone dystrophy with supernormal rod response (CDSRR; OMIM # 610356) is a rare inherited retinal disorder related to mutations in the potassium voltage-gated channel modifier subfamily V member 2 (KCNV2) gene. CDSRR can be inherited due to autosomal recessive form mutations in the KCNV2 gene on chromosome 9 [1, 2]. Associated symptoms appear in the first two decades of life [3]. Furthermore, patients with CDSRR may experience nyctalopia, photophobia, and mild to moderate myopia until the second decade of life as a result of changes in the macular retinal epithelial and the retinal structural and functional disorders. Cases of CDSRR are most often reported in consanguineous marriages, which may increase the risk of recessive diseases. Since CDSRR has been described as a rare retinal disorder with autosomal recessive inheritance, it is also associated with suprathreshold rod responses and significant reductions in cone-rod electrophysiology (ERG) under high-stimulus conditions [4, 5, 6]. CDSRR childhood is characterized by visual acuity reaching 20/100 or less in the second decade of life. Most patients experience myopia and late onset of night blindness. It has been noted that some patients with CDSRR develop a normal fundus with parafoveal/foveal atrophy [7]. The number of people affected by CDSRR is estimated to be around 1 in 1,000,000, effectively classifying the disease as rare. The availability of electroretinography (ERG) and genetic testing allows accurate and early diagnosis of CDSSR and facilitates the development of treatments for this disease [8].

CDSRR is caused by mutations in the KCNV2 gene, which encodes the Kv8.2 subunit of the voltage-gated potassium channel. As with other autosomal recessive diseases, CDSRR is a good candidate for gene therapy [9], the main strategy of which is to overexpress the wild-type copy of the gene in retinal neurons. Multiple models can be tested to apply optimized gene therapy to the retina; healthy human retinas isolated from cadaver eyes and healthy wild-type primate retinas are also considered good approximations. Furthermore, creating suitable non-human primate models poses ethical and resource challenges, but some naturally occurring mutations related to retinal degeneration have been identified in existing NHP populations. Additionally, various inducible models have been attempted using methods like gene delivery and chemical injections. The field of ocular gene and cell therapies is rapidly advancing, but there is a critical need for effective preclinical models to test new therapies and understand disease mechanisms. While existing rodent models are useful for initial studies, they fail to replicate essential anatomical features of human diseases, such as macula and fovea structure. Gene transfer using adeno-associated viruses (AAVs), shows promise for treating inherited retinal diseases, as demonstrated in animal models. Studies investigated the efficiency of AAV in primate retinas through subretinal injections across various retinal regions. Results demonstrated rapid transgene expression within a few weeks, lasting up long, while maintaining overall retinal function [10, 11, 12].

A new alternative is the use of retinal tissue derived from stem cells. In this scenario, induced pluripotent stem cells (iPSCs) are derived from the blood cells of patients. iPSCs can be expanded and frozen for numerous studies. Retinal tissues (organoids) are then differentiated from iPSCs using a 3D strategy in large quantities. Studies have shown that these organoids can be effectively used to study AAV targeting host cells, as well as for disease modeling and drug discovery. hiPSCs hold significant potential for studying retinal development, diseases, and therapies. Gene modulation in hiPSCs is crucial, and AAV vectors have shown promise for gene delivery in retinal organoids derived from hiPSCs. Several studies investigated the efficiency of various recombinant and engineered AAVs in transducing hiPSC-derived retinal pigment epithelium (RPE) cells and retinal organoids, focusing on cell-surface receptor availability and time factors. Synthetic AAV variants demonstrated high transduction efficiency when applied at retinal organoids, ensuring prolonged expression without affecting cell viability [13, 14].

In modern practice, gene therapy involves the functional replacement of a dysfunctional gene that does not produce a functional protein with a wild-type copy that restores function. Gene therapy is a promising approach for the treatment of inherited and common complex retinal diseases, and preclinical and clinical studies have supported the use of AAVs as a safe and effective means of gene delivery [12].

In this dissertation, we aimed to develop three-dimensional retinal organoid models derived from hiPSC lines obtained from patients with CDSRR. Our objective was to establish that these retinal organoids serve as an effective platform for evaluating AAV strategies in gene therapy studies for CDSRR. Furthermore, we sought to demonstrate that retinal organoids can accurately replicate the morphological and functional characteristics of a healthy human retina.

Purpose of the work and tasks

The objective of this dissertation is to establish a patient-derived iPSC-based retinal organoids as a platform for the screening of AAV gene therapies aimed at treating inherited retinal degenerations (IRDs). To achieve this goal, we set the following objectives:

1. A comprehensive description of the clinical progression and associated molecular findings in patients exhibiting CDSRR linked to recessive mutations in the KCNV2 gene.

2. The establishment of human KCNV2-mutant iPSC lines through the reprogramming of blood cells using Sendai virus, followed by the characterization, expansion, and preservation of these cell lines to confirm their pluripotent nature.

3. The differentiation of KCNV2-hiPSCs into retinal organoids, with an initial focus on assessing early retinal differentiation to validate the feasibility of this approach.

4. An investigation of the complete differentiation of retinal organoids from KCNV2-hiPSCs, aiming

to generate retinal tissue encompassing photoreceptor and bipolar cell populations.

5. An analysis of KCNV2 expression within iPSC-derived human retinal organoids, with comparisons made to cadaveric healthy human retinal tissue.

6. The characterization of functional deficits in KCNV2-hiPSC-derived retinal organoids through the application of whole-cell patch clamp techniques.

7. The identification of the optimal AAV vectors for targeting human photoreceptors, utilizing cadaveric retinal samples, mouse models, and hiPSC-derived retinal organoids.

8. An exploration of the delivery and expression of a codon-optimized copy of the KCNV2 gene facilitated by available AAV vectors within KCNV2-hiPSC-derived retinal organoids.

Scientific novelty

The findings presented in this dissertation are novel, with many results being reported for the first time. An optimized protocol for the generation of human retinal organoids was developed, and these organoids were thoroughly characterized using various methodologies to confirm the presence of multiple cell types characteristic of the retina, including photoreceptors, bipolar cells, ganglion cells, and retinal pigment epithelial cells. Furthermore, a protocol for the functional electrophysiological analysis of individual photoreceptor-like cells within the retinal organoids was established, utilizing the patch clamp technique. iPSC lines were derived from two patients exhibiting a compound heterozygous mutation in the KCNV2 gene. One of these patients harbored a de novo mutation in the KCNV2 gene (c.1109dup). Subsequently, the iPSC lines were differentiated into retinal organoids. A comprehensive molecular and functional comparison of these organoids was conducted. Additionally, a healthy copy of the KCNV2 gene was successfully introduced into knockout retinal organoids using AAV8, incorporating green fluorescent protein (GFP) as a reporter gene.

Theoretical and practical significance

Cone dystrophy with supernormal rod response (CDSRR) is a form of retinal dystrophy characterized by a spectrum of visual symptoms that typically manifest during childhood and progressively worsen over time. The hallmark symptoms of CDSRR are indicative of dysfunction in the cone photoreceptors, with a lesser involvement of rod photoreceptors, leading to specific types of visual impairment. Early manifestations of CDSRR often include a marked reduction in visual acuity, which is defined as a significant decline in the sharpness and clarity of vision, particularly affecting the central visual field. CDSRR is recognized as a serious visual impairment due to its poor prognosis and the associated decline in patients' quality of life over time, exacerbated by the absence of effective treatments to halt or slow the progression of visual loss. Available interventions are limited to optical aids and lenses that may only partially alleviate the progression of visual impairment. Furthermore, the

poor prognosis of CDSRR often diminishes the likelihood of recovery, given the current lack of curative therapies. To date, there has been insufficient extensive research into the natural history and pathogenesis of CDSRR that fully elucidates the progression of the condition. Nevertheless, several studies have documented the natural progression of central vision loss in affected individuals.

The outcomes of this study may enhance understanding of CDSRR, particularly in the context of mutations in the KCNV2 gene. CDSRR emerges as an ideal candidate for AAV gene therapy based on several criteria, including the presence of recessive mutations, the small size of the KCNV2 coding region, the monogenic nature of the disorder, and the timing of disease onset, which collectively inform the therapeutic window for potential interventions. Moreover, this research paves the way for the development of gene therapy, thereby improving treatment options for affected patients. The thesis further outlines practical recommendations for gene delivery methods targeting CDSRR, utilizing iPSC-derived retinal organoids as a platform for retinopathy gene therapy, alongside the selection of appropriate vectors and assay protocols in comparison to alternative animal models. Overall, these findings are crucial for advancing new therapeutic strategies, enhancing the treatment of monogenic diseases, and validating retinal organoids as a viable model for the investigation and visualization of CDSRR in scientific research.

Methodology and research methods

As part of the study, AAV serotypes were tested ex vivo on human retinal organoids and cultured for seven days under defined conditions. Human retinal explants were obtained from cadaver eyes in collaboration with the S.N. Fedorov National Ophthalmology Medical Research Center "Eye Microsurgery", Moscow. Retinal explant transduction was assessed using immunohistochemistry analysis and flow cytometry. Mouse models (C57BL/6) were used to test AAV vectors in vivo by subretinal injection. The retina was isolated after AAV administration after a month of injections. Therefore, the results were analyzed using immunohistochemical staining. As part of the work on task iPSC lines were obtained from patients with inherited retinal disease (CDSRR). The first patient was observed at Pediatric city clinical hospital named for Z.A. Bashlyaevoy, Moscow, while the second patient was observed at S. Fyodorov "Eye Microsurgery" Federal State Institution St. Petersburg Branch, St. Petersburg. Venous blood samples were obtained from patients and mononuclear cells were isolated. Mononuclear cells were reprogrammed using a commercial kit based on the Sendai virus expressing four Yamanaka factors. After transduction, colonies with morphology consistent with hESC were expanded, partially frozen, and partially used to characterize these iPSC lines.

Work has also been carried out to optimize the protocol for obtaining retinal-like 3D cell models (organoids). Protocols published by various scientific groups were reviewed. We chose the Sasai protocol with some modifications as a basis. We refined this protocol and determined the optimal

concentration of used Matrigel. To confirm the formation of various types of retinal cells during the differentiation process, immunocytochemical analysis, flow cytometry and quantitative real-time PCR of organoids was carried out at different stages of differentiation. To characterize the function of retinal organoids, a whole-cell patch clamp method was applied to record the activity of photoreceptor-like cells. Patch clamp experiments carried out in the Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences, Moscow. AAV vectors were generated by transfection of HEK-293T cells, purified by ultracentrifugation, and titrated by qPCR. KCNV2 gene expression was assessed using immunohistochemistry and quantitative real-time PCR.

Statements to be defended

1. A novel mutation in the KCNV2 gene associated with CDSRR was identified.

2. AAV tropism and potency were assessed in retinal models to show that AAV8 has high tropism in photoreceptor cells.

3. A differentiation protocol for retinal organoids derived from induced iPSCs was developed, along with a comprehensive characterization of these organoids.

4. KCNV2 expression levels were found to be lower in knockout retinal organoids compared to those in human retina.

5. Functional characterization revealed that the photoreceptor-like cells in KCNV2-knockout retinal organoids exhibited a reduced current response compared to those in healthy organoids.

6. A functional copy of the KCNV2 gene was successfully introduced into knockout retinal organoids using AAV8.

Author's contribution

The study was conducted on the basis of the Federal State Autonomous Educational Institution of Higher Education "Moscow Institute of Physics and Technology (National Research University)" in the period from 2020 to 2024. The author undertook an independent analysis of contemporary domestic and international literature relevant to the research topic. The development and refinement of the comprehensive study were carried out independently. The author participated in all experimental investigations, both in vitro and in vivo. The applicant actively conducted data analysis, authored manuscripts, compiled and obtained results, and contributed to the writing and publication of scientific articles and conference proceedings. The findings presented in this thesis are the result of four years of research conducted by the author as a PhD student and junior researcher in the Genome Engineering Laboratory at the Moscow Institute of Physics and Technology (MIPT).

Structure and scope of the dissertation

The dissertation consists of 155 pages of typewritten text and is organized into several sections:

Introduction, Literature Review, Materials and Methods, Results, Discussion, Conclusions and Outlook,

and References. It includes 8 tables and 50 figures, and the reference list contains 204 publications.

Approbation of the work

Based on the findings of the dissertation, 3 conference abstracts were created. The research was

presented at the following conferences:

1. Oral Presentation in the 64th All-Russian Scientific Conference of the Moscow Institute of Physics and Technology (MIPT) (2022, Dolgoprudny, Russia).

2. Oral presentation in Annual conference of the Syrian Association of Pathology, Syrian Molecular Pathology Assembly (2nd SMGA) (2022, Damascus, Syria).

3. Oral presentation in III International Scientific Conference "Stem Cell Bio - 2023: Translational Medicine - a Spectrum of Possibilities" (2023, St. Petersburg, Russia).

In addition, 3 works were published related to the theme, all of them in journals indexed in Web

of Science and Scopus. The work was tested at the following:

1. A. Alsalloum; O. Mityaeva; E. Kegele; E. Khavina; P. Volchkov, Generation of two human induced pluripotent stem cell lines (ABi001-A and ABi002-A) from cone dystrophy with supernormal rod response patients caused by KCNV2 mutation, Stem Cell Research. 2023, doi: 10.1016/j.scr.2023.103099.

2. A. Alsalloum; K. Shefer; P. Bogdanov; N. Mingaleva; A. Kim; S. Feoktistova; O. Mityaeva; P. Volchkov, Establishment of a human induced pluripotent stem cell line (ABi004-A) carrying a compound heterozygous mutation in the KCNV2 gene, Stem Cell Research, 2024, doi: 10.1016/j.scr.2024.

3. A. Alsalloum; I. Mosin; K. Shefer; N. Mingaleva; A. Kim; S. Feoktistova; B. Malyugin; E. Boiko; S. Sultanov; O. Mityaeva; P. Volchkov, Novel and Previously Known Mutations of the KCNV2 Gene Cause Various Variants of the Clinical Course of Cone Dystrophy with Supernormal Rod Response in Children, Journal of Clinical Medicine, 2024, doi: 10.3390/jcm13164592.

Заключение диссертации по теме «Другие cпециальности», Алсаллум Алмакдад

MAIN RESULTS AND OUTLOOK

I. Clinical and molecular investigations were obtained from two patients with compound heterozygous mutations in the KCNV2 gene related to CDSRR. Analysis of the KCNV2 gene in the second patient revealed a de novo mutation (c.1109dup).

II. The human retinal explant model was successfully tested using various natural serotypes and synthetic AAV, finding that the AAV8 serotype efficiently transduced photoreceptors and bipolar cells. Subretinal injection in mouse models showed high efficiency and selectivity of AAV5 and AAV8 vectors in transducing photoreceptor cells.

III. iPSC lines were obtained and characterized from the first and second patient. The cell lines obtained from the first patient and his mother were registered under the unique identifiers ABi002-A and ABi001-A, respectively. ABi004-A was the identifier of a cell line obtained from the second patient.

IV. hESC-derived retinal organoids were successfully generated using an optimized differentiation protocol. The organoids were characterized at early, middle and late stages to confirm their morphological similarity to healthy human retina by expressing various retinal cell markers.

V. Morphological characterization of retinal organoids derived from the ABi001-A, ABi002-A, and ABi004-A lines was successfully obtained for further use as a platform for AAV-based CDSRR gene therapy.

VI. Retinal organoids derived from ABi002-A showed an absence of KCNV2 protein by immunohistochemical analysis compared to expression of the KCNV2 product in hESCs and ABi001-A-derived retinal organoids.

VII. ABi002-A-derived retinal organoids revealed an association between a compound heterozygous mutation pattern in the KCNV2 gene and functional response impairment in photoreceptor-like cells, particularly in current amplitude responses compared with ABi001-A-derived retinal organoids using patch clamp.

VIII. ABi002-A-derived retinal organoids were transduced using AAV8 under the rhodopsin kinase promoter. Immunohistochemical analysis confirms the delivery of the codon-optimized KCNV2 gene into the retinal organoids.

Список литературы диссертационного исследования кандидат наук Алсаллум Алмакдад, 2024 год

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