Разработка генной терапии врожденной гиперплазии надпочечников с использованием векторов AAV и редактирования генома, опосредованного CRISPR/Cas9 / Development of gene therapy for congenital adrenal hyperplasia using AAV vectors and CRISPR/Cas9-mediated genome editing тема диссертации и автореферата по ВАК РФ 00.00.00, кандидат наук Сакр Навар

• Сакр Навар
• кандидат наук
• 2025

Оглавление диссертации кандидат наук Сакр Навар

Table of contents

Abstract

Introduction

Aim and objectives of the study

Relevance of the work

Scientific novelty

Theoretical and Practical Significance

Author's contribution

Statements to be defended

Approbation of the thesis

Publications

Structure and size of the work

Chapter 1. Literature review

1.1 Adrenal gland structure and function

1.2 Congenital adrenal hyperplasia

1.2.1 Pathophysiology of CAH

1.2.2 Standard management and treatment protocols for CAH

1.3 Gene therapy

1.3.1 Adeno-associated viral (AAV) vectors

1.3.2 Studies of gene therapy for CAH

1.3.3 Limitations of gene-addition therapies in CAH

1.4 Genome editing

1.4.1 CRISPR/Cas9 system

1.4.2 Therapeutic applications of the CRISPR/Cas9

1.4.3 Safety and ethical considerations

Chapter 2. Materials and methods

2.1 Plasmids

2.2 Restriction enzyme digestion

2.3 DNA ligation

2.4 Plasmid propagation and purification

2.5 Cell Culture

2.6 Nucleic acids extraction

2.7 Polymerase chain reaction (PCR)

2.8 Vector genome copy numbers per cell (VGC/cell)

2.9 Digital PCR (dPCR)

2.10 AAV production

2.11 AAV titration

2.12 Transfection and transduction

2.13 B16-Cas9 cells production

2.14 CRISPR-Cas9 editing efficiency

2.15 Genotyping

2.16 Animal procedures

2.17 Immunofluorescence assays

2.18 Autofluorescence characterization and treatment

2.19 Steroid hormone assay

2.20 Statistical analysis

Chapter 3. Results

3.1 Development and validation of vectors for CAH gene therapy

3.1.1 Rosa26 locus targeting using the CRISPR/Cas9 system

3.1.2 Construction and validation of plasmids for Cyp21al expression and targeted knock-in

3.1.3 Functional evaluation of genetic constructs

3.1.4 Assessment of knock-in efficiency via CRISPR/Cas9-mediated homology-dependent and independent DNA repair

3.1.5 AAV-based CRISPR/Cas9-mediated knock-in of the Cyp21a1 expression cassette at the Rosa26 locus

3.2 Optimization of in vivo protocols for gene therapy development

3.2.1 Evaluation of the intra-adrenal administration route for gene delivery to the adrenal glands77

3.2.2 Gene expression and CRISPR/Cas9-mediated knock-in following intra-adrenal delivery

3.2.3 Gene delivery to adrenal glands via systemic AAV administration

3.2.4 Tropism of AAV serotypes for the adrenal glands following systemic injection

3.2.5 Characterizing and quenching autofluorescence in mouse adrenal Cortex tissue

3.2.6 Establishing the protocol for obtaining Cyp21a1-- mice

3.3 Therapeutic outcomes of gene therapies in Cyp21a1-/- mice

3.3.1 AAV-DJ-mediated expression and CRISPR/Cas9-mediated knock-in in the adrenal glands94

3.3.2 Long-term study design for evaluating AAV-mediated gene therapy and CRISPR/Cas9-based gene editing therapy for CAH in Cyp21a1-/- mice

3.3.3 Evaluation of vector genome and mRNA expression profiles in Cyp21a1-/- mouse tissues

3.3.4 Immunofluorescence analysis of AAV-mediated expression in Cyp21a1-/- adrenal and liver tissues

3.3.5 Evaluation of steroid hormone profiles

3.3.6 Evaluation of body weight changes

3.3.7 Survival of Cyp21a1mice after treatment

3.4 Evaluation of optimized genome-editing therapy in Cyp21a1-- mice

3.4.1 Dose selection and study design

3.4.2 Analysis of vector genome copy number per cell in adrenal glands and liver

3.4.3 Knock-in frequency in the adrenal glands and liver

3.4.4 Immunofluorescence analysis of AAV-mediated expression in Cyp21a1-/- adrenal tissue134

3.4.5 Assessment of serum steroid hormone dynamics in Cyp21a1-/- mice following treatment135

3.4.6 Evaluation of body weight and survival in Cyp21a1-/- mice after treatment

Chapter 4. Discussion

Conclusion

Main results and the outlook

Abbreviations

Acknowledgments

References

Supplementary Materials

List of Figures

Figure 1.1 Congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Figure 1.2 Gene therapy using recombinant AAV vectors

Figure 1.3 Schematic of the adrenal cortex renewal

Figure 1.4 Programmable gene editing using ZFNs, TALENs, and CRISPR/Cas9

Figure 1.5 The mechanism of non-homologous end joining and Homology-directed repair

Figure 1.6 Genome editing with programmable nucleases

Figure 3.1 CRISPR/Cas9 genome-editing efficiency at the Rosa26 locus

Figure 3.2 Schematic representations of plasmid constructs for Cyp21a1 expression and CRISPR/Cas9-

mediated knock-in

Figure 3.3 Restriction digestion and sequencing analyses of plasmid constructs

Figure 3.4 Evaluation of plasmid packaging into the AAV-2 vector

Figure 3.5 Optimization of B16 cell transfection

Figure 3.6 Evaluation of plasmid-mediated expression of 21-hydroxylase, EGFP, and Cas9

Figure 3.7 Efficiency of CRISPR/Cas9-mediated knock-in via NHEJ and HDR repair mechanisms

Figure 3.8 AAV-CRISPR/Cas9-mediated genome editing at the Rosa26 locus

Figure 3.9 Distribution of AAV serotypes in adrenal glands and liver after intra-adrenal injection

Figure 3.10 Impact of local AAV injection on survival and adrenal hormone concentrations in mice

Figure 3.11 Gene expression and CRISPR/Cas9 genome editing following AAV intra-adrenal

administration

Figure 3.12 Systemic administration of AAV for gene delivery to the adrenal glands

Figure 3.13 AAV-mediated transgene expression in adrenal glands and liver after systemic injection

Figure 3.14 Autofluorescence (AF) emission spectra of mouse adrenal cortex tissue sections

Figure 3.15 The effect of different tissue treatments on green wavelength autofluorescence

Figure 3.16 Reduction of AF from pigment-laden cells in the adrenal tissue of aged mice

Figure 3.17 IF staining of adrenal tissue sections treated with TrueBlack™ or MaxBlock™

Figure 3.18 EGFP detection in fixed frozen adrenal tissue sections treated with TrueBlack™

Figure 3.19 PCR-RFLP assay for genotyping of H-2aw18 mice

Figure 3.20 TaqMan-probe-based multiplex real-time qPCR assay for Cyp21a1 genotyping

Figure 3.21 Efficient gene delivery and targeted knock-in in the adrenal glands of Cyp21a1-/- mice following gene delivery using AAV-DJ

Figure 3.22 Study design for evaluating CAH genome-editing therapy in Cyp21a1-/- mice

Figure 3.23 The VGC/cell in the adrenal glands at different time points post AAV injection

Figure 3.24 AAV-mediated mRNA expression in the adrenal glands at different time points post AAV

injection

Figure 3.25 The VGC/cell in the liver at different time points post AAV injection

Figure 3.26 AAV-mediated mRNA expression in the liver at different time points post AAV injection

Figure 3.27 The VGC/cell in the kidneys and gonads at different time points post AAV injection

Figure 3.28 The VGC/cell in the heart and brain at different time points post AAV injection

Figure 3.29 Summary of VGC/cell in different mouse tissues

Figure 3.30 Immunofluorescence staining in Cyp21a1-/- mouse adrenal tissue 4 weeks post-injection

Figure 3.31 EGFP fluorescence in Cyp21a1-/- mouse liver tissue 4 weeks post-injection

Figure 3.32 Immunofluorescence staining in Cyp21a1-/- mouse adrenal tissue 8 weeks post-injection

Figure 3.33 EGFP fluorescence in Cyp21a1-/- mouse liver tissue 8 weeks post-injection

Figure 3.34 Immunofluorescence staining in Cyp21a1-/- mouse adrenal tissue 16 weeks post-injection

Figure 3.35 EGFP fluorescence in Cyp21a1-/- mouse liver tissue 16 weeks post-injection

Figure 3.36 EGFP fluorescence in Cyp21a1-/- mouse liver tissue 30 weeks post-injection

Figure 3.37 Immunofluorescence staining in Cyp21a1-/- mouse adrenal tissue 4 weeks post-injection

Figure 3.38 Serum concentrations of 11-deoxycorticosterone in mouse serum

Figure 3.39 Serum concentrations of corticosterone in mouse serum

Figure 3.40 Serum concentrations of progesterone in mouse serum

Figure 3.41 Serum concentrations of aldosterone and 18-hydroxycorticosterone in mouse serum

Figure 3.42 Body weight gain in Cyp21a1-- mice after treatment

Figure 3.43 Evaluation of survival rates in Cyp21a1-/- mice post-treatment

Figure 3.44 Dose selection and study design for the evaluation of optimized genome-editing therapy in

Cyp21a1-/- mice. (A)

Figure 3.45 VGC/cell in adrenal glands and liver of Cyp21a1-/- mice at week 28 post-injection

Figure 3.46 Knock-in frequency in adrenal glands and liver of Cyp21a1-/- mice at 28 weeks postinjection

Figure 3.47 Immunofluorescence staining of adrenal tissue from Cyp21a1-/- mice 28 weeks postinjection

Figure 3.48 Serum concentrations of steroid hormones in Cyp21a1-/- mice after treatment

Figure 3.49 Body weight and survival of Cyp21a1-/- mice after treatment

List of Tables

Table 1. 1 Summary of preclinical gene therapy studies on a mouse model of CAH

Table 2.1 Plasmids used in this study

Table 2. 2 List of primers and probes for real-time PCR and digital PCR

Table 2.3 List of genotyping primers and probes for multiplex real-time PCR

Table 2.4 List of antibodies used in this study

Supplementary Table 1.1 Wild-type and H-2aw18 Cyp21a1 sequences

Supplementary Table 1.2 The mean VGC/cell (SD) of AAV-Cyp21 in different mouse tissues at different

time points following injection with AAV-DJ-Cyp21 and AAV-DJ-Cas9

Supplementary Table 1.3 The mean VGC/cell (SD) of AAV-Cyp21 in different mouse tissues at different

time points following injection with AAV-DJ-Cyp21

Supplementary Table 1.4 Serum concentrations of 11-deoxycorticosterone in mice from different

experimental groups at various time points post-injection

Supplementary Table 1.5 Serum concentrations of corticosterone in mice from different experimental

groups at various time points post-injection

Supplementary Table 1.6 Serum concentrations of progesterone in mice from different experimental

groups at various time points post-injection

Supplementary Table 1.7 Serum concentrations of aldosterone in mice from different experimental groups

at various time points post-injection

Supplementary Table 1.8 Serum concentrations of 18-hydroxycorticosterone in mice from different experimental groups at various time points post-injection

Введение диссертации (часть автореферата) на тему «Разработка генной терапии врожденной гиперплазии надпочечников с использованием векторов AAV и редактирования генома, опосредованного CRISPR/Cas9 / Development of gene therapy for congenital adrenal hyperplasia using AAV vectors and CRISPR/Cas9-mediated genome editing»

Abstract

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is an inherited disorder characterized by impaired Cortisol and aldosterone synthesis, leading to significant metabolic complications and life-threatening adrenal crises. The current hormone replacement therapy fails to mimic the endogenous hormones' circadian rhythm and is associated with long-term complications due to glucocorticoid overexposure. Gene therapy offers an alternative approach, targeting the underlying genetic defect to restore steroid hormone production. This thesis presents a proof-of-concept study investigating CRISPR/Cas9-mediated genome-editing therapy in a Cyp21a1-/- mouse model of CAH. Novel genetic constructs were developed to mediate Cyp21a1 expression and to enable site-specific knock-in at the Rosa26 safe harbor locus in the mouse genome. The efficiency of homology-directed repair (HDR) and non-homologous end joining (NHEJ) was evaluated for CRISPR/Cas9-mediated gene knock-in. NHEJ was more efficient than HDR in mediating the integration of the Cyp21a1 expression cassette into the Rosa26 locus. The local and systemic administration routes of adeno-associated virus (AAV) vectors for gene delivery to the adrenal glands were evaluated. Intra-adrenal injection did not enhance the adrenal specificity of AAV transduction and failed to achieve sufficient transgene expression in the adrenal cortex. In contrast, systemic administration resulted in higher vector genome copy numbers per cell (VGC/cell) in the adrenal glands compared to local delivery. Strong transgene expression was achieved in the adrenal glands and liver following systemic injection of the synthetic AAV-DJ serotype. AAV-based CRISPR/Cas9-mediated knock-in via the NHEJ pathway enabled stable targeted integration of the Cyp21a1 expression cassette into the genomes of adrenal and hepatic cells, detectable for at least 28 weeks post-injection. Genome-editing therapy resulted in durable therapeutic effects. Treated mice exhibited stable restoration of 11-deoxycorticosterone concentrations, prolonged improvement in body weight, and 100% survival over 28 weeks after AAV injection. This thesis demonstrates the feasibility of CRISPR/Cas9-mediated genome editing as a therapeutic approach for CAH and highlights its potential to achieve sustained correction of steroidogenesis. While further studies are necessary to optimize delivery methods, improve genome-editing efficiency, and address concerns regarding the long-term safety of genome-editing approaches, these findings provide a strong foundation for advancing CRISPR/Cas-based therapies toward clinical translation.

Introduction

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders that affect adrenal steroidogenesis. The most common form, accounting for over 90% of cases, is 21-hydroxylase deficiency (21-OHD), which results from mutations in the human CYP21A2 gene [1]. The 21-hydroxylase enzyme plays a critical role in the biosynthesis of cortisol and aldosterone. The absence or reduction of 21-hydroxylase activity leads to the inability to produce these hormones and an overproduction of adrenal androgens [1, 2]. The terms CAH and 21-OHD will be used interchangeably in this thesis.

Globally, CAH due to 21-OHD occurs in approximately 1 in 14,000 to 1 in 18,000 live births, with significant regional and ethnic variations [3]. The disorder presents a spectrum of clinical phenotypes based on the level of residual 21-hydroxylase activity. In severe, classic salt-wasting forms of CAH, patients face life-threatening adrenal crises due to deficiencies in cortisol and aldosterone, alongside virilization caused by excess androgen production [4, 5]. The deficiency in cortisol leads to a disruption in the feedback loop that regulates the hypothalamic-pituitary-adrenal axis. In the absence of sufficient cortisol, adrenocorticotropic hormone (ACTH) is continuously released from the pituitary gland, leading to adrenal hyperplasia and excessive production of androgens [3].

Despite advancements in early diagnosis and treatment, CAH continues to be associated with significant morbidity and mortality [6, 7]. The standard treatment, glucocorticoid and mineralocorticoid replacement therapy introduced in the 1950s, has dramatically improved survival rates but poses considerable challenges. Current therapies fail to replicate the natural circadian rhythm of cortisol production, often requiring supraphysiological doses, which contribute to long-term complications such as growth suppression, obesity, osteoporosis, and fertility problems [5, 8-11]. Additionally, hormonal replacement therapy addresses only the symptoms without correcting the underlying genetic defect, leaving patients reliant on lifelong medication and vulnerable to adrenal crises, particularly during stress or illness. These limitations underscore the need for more targeted and durable therapeutic approaches to restore adrenal hormone production and reduce the long-term adverse effects of current treatments [12, 13].

In recent years, gene therapy has emerged as a promising approach to addressing the underlying cause of monogenic diseases. However, early gene therapy studies for CAH treatment have encountered significant limitations, particularly concerning the durability of therapeutic effects [12, 14, 15]. The rapid cellular turnover of the adrenal cortex presents a challenge for current adeno-associated virus (AAV)-mediated gene-addition strategies [16, 17]. Gene-addition strategies rely primarily on adeno-associated

virus (AAV) episomal expression, which gradually declines over time in regenerative tissues like the adrenal glands. This decline limits the long-term efficacy of these therapies [12, 15].

The lack of an optimal AAV vector with strong tropism for adrenal tissue and the ability to sustain long-term gene expression has limited the success of gene therapy approaches for CAH. The use of novel AAV vectors with enhanced adrenal gland tropism could significantly improve therapeutic outcomes by enabling more efficient and sustained gene delivery to the target tissue.

The emergence of CRISPR/Cas9 technology has revolutionized gene therapy, offering a precise and versatile tool for targeted genome modification. Initially discovered as part of the bacterial immune defense system, CRISPR/Cas9 has been adapted into a powerful technology for precise genetic engineering [18, 19]. This approach holds great promise for gene therapy, particularly for correcting monogenic disorders like CAH, where permanent genetic correction could eliminate the need for lifelong treatments [12].

More than 100 distinct mutations have been identified in the human CYP21A2 gene, including deletions, point mutations, and insertions [20]. Given the complexity and diversity of these mutations, designing specific therapies tailored to individual mutations would be time-consuming, resource-intensive, and unlikely to provide a comprehensive solution for the entire CAH population. Additionally, most CAH patients are compound heterozygotes, carrying two different disease-causing mutations in the CYP21A2 gene, further complicating the development of mutation-specific therapies [21, 22].

Instead, the knock-in of a fully functional copy of the 21-hydroxylase gene via CRISPR/Cas9-mediated genome editing offers a more efficient and broadly applicable therapeutic strategy for addressing the full spectrum of CAH mutations. By integrating the functional gene into the genome of adrenal cortex stem or progenitor cells, the therapeutic gene becomes a permanent part of the genome, allowing corrected cells to proliferate and continuously produce the necessary steroid hormones. This approach eliminates the reliance on transient gene expression, enhances the durability of therapeutic effects, and could ultimately reduce or even eliminate the need for lifelong hormone replacement therapy.

Therefore, addressing the limitations of both hormone replacement therapy and current gene therapy approaches is crucial. There is an urgent need for innovative therapeutic strategies that not only restore physiological cortisol production but also offer durable solutions for managing CAH, significantly reducing patient reliance on lifelong steroid replacement therapy and minimizing associated complications.

Aim and objectives of the study

The aim of this study is to develop gene therapy approaches for congenital adrenal hyperplasia due to 21-hydroxylase deficiency using AAV vectors and CRISPR/Cas9-mediated genome editing.

To achieve this aim, the following objectives were defined:

1. Construction of gene expression cassettes and assembly of AAV viral vectors for CAH genome-editing therapy.

2. Optimization of AAV administration routes for targeted and efficient gene delivery to the mouse adrenal glands.

3. Evaluation of AAV serotype tropism for the mouse adrenal glands.

4. Assessment of CRISPR/Cas9-mediated site-specific gene knock-in in the mouse adrenal glands.

5. Evaluation of long-term therapeutic outcomes of AAV-based CRISPR/Cas9-mediated genome-editing therapy in the Cyp21a1-/- mouse model.

In this study, genetic constructs were developed to enable expression of the Cyp21a1 gene and to allow its CRISPR/Cas9-mediated knock-in at the safe harbor Rosa26 locus in the mouse genome. The efficiency of homology-directed repair (HDR) and non-homologous end joining (NHEJ) DNA repair mechanisms in mediating gene knock-in was evaluated in cell lines. The administration routes of AAV vectors —both local intra-adrenal and systemic injections —were compared to optimize AAV delivery to the adrenal cortex. To determine the most effective AAV serotype for targeted gene delivery to the adrenal glands, the tropism of various wild-type (WT) and synthetic AAV serotypes was evaluated following both local and systemic administration. The AAV-mediated expression of the Cyp21a1 gene in cell lines and mouse tissues was evaluated to assess the efficiency of gene delivery and expression in various biological systems. Additionally, the knock-in of the Cyp21a1 gene into the target Rosa26 locus, following delivery of the CRISPR/Cas9 system components and the knock-in donor sequence using a dual AAV vector approach, was assessed both in vitro and in vivo.

In this study, Cyp21a1-/- mice were used as a model for CAH due to 21-OHD to evaluate the efficacy of the therapeutic approach. Cyp21a1-/- mice exhibit a phenotype similar to human CAH, including impaired steroidogenesis and adrenal hyperplasia. The biodistribution of AAV vectors across several tissues, including the adrenal glands, liver, kidneys, heart, brain, and gonads, was analyzed to evaluate tissue-specific targeting and non-target tissue transduction. Additionally, the dynamics of vector genome

copy numbers and gene expression in the adrenal glands and liver were analyzed at multiple time points to assess the persistence of vector genomes and the durability of gene expression in these tissues. Furthermore, comprehensive steroid hormone profiles were analyzed to evaluate the therapeutic impact of the genome-editing strategy on restoring steroidogenesis. In addition, weight gain and survival rates were monitored as indicators of treatment effectiveness, reflecting overall health and physiological improvements in treated animals.

This research represents a pioneering effort to address critical challenges in the treatment of CAH due to 21-hydroxylase deficiency. To our knowledge, this proof-of-concept study is the first to apply CRISPR/Cas9-based genome editing for the treatment of CAH in an animal model. By using AAV-based CRISPR/Cas9-mediated knock-in of the Cyp21a1 gene into the adrenal gland cells' genome, this study aims to achieve prolonged therapeutic effects and offer a more durable, long-term solution to the genetic disorder. The study demonstrated that the NHEJ DNA repair mechanism mediates site-specific gene integration in the mouse adrenal gland genome. Another essential contribution of this study is its focus on improving gene delivery to the adrenal glands using engineered AAV capsids. To our knowledge, this study is the first to demonstrate the strong tropism of the synthetic AAV-DJ serotype for the adrenal cortex, highlighting its potential to enhance gene delivery efficiency despite its strong tropism for liver tissue. These findings have important implications not only for CAH but also for other genetic adrenal disorders, where targeted delivery to adrenal tissue is critical for effective therapy. Furthermore, this research offers a comprehensive evaluation of AAV administration routes for efficient gene delivery to the adrenal glands.

The evaluation of the long-term therapeutic potential of the genome-editing approach is a central focus of this study. By demonstrating sustained gene expression, improved steroid hormone profiles, and improved survival rates in Cyp21a1-/- mice, the study demonstrates the potential to reduce or even eliminate the need for lifelong hormone replacement therapy. The findings are applicable to the development of gene therapies for other adrenal insufficiencies and disorders and contribute to the optimization of AAV-based CRISPR/Cas9 genome editing in highly regenerative tissues such as the adrenal cortex.

Relevance of the work

CAH due to 21-OHD is one of the most common autosomal recessive disorders, affecting

approximately 1 in 14,000 to 18,000 live births worldwide. 21-OHD disrupts adrenal steroidogenesis,

resulting in cortisol and aldosterone deficiencies and excessive adrenal androgen production. The clinical

consequences range from hyperandrogenism and virilization to life-threatening adrenal crises. Despite

advancements in neonatal screening and hormone replacement therapies, the global burden of CAH

13

remains significant, with considerable morbidity and mortality, particularly in regions where screening programs are less accessible.

The standard treatment for CAH involves lifelong hormone replacement therapy with glucocorticoids and mineralocorticoids to compensate for cortisol and aldosterone deficiencies. However, these therapies do not address the underlying genetic defect and often require supraphysiological doses to suppress androgen excess. The prolonged use of such high doses leads to serious side effects, including growth suppression, obesity, metabolic disorders, and osteoporosis. Moreover, hormone replacement therapy fails to restore normal physiological functions, such as the natural circadian rhythm of cortisol production, and leaves patients susceptible to adrenal crises during illness or stress. These limitations highlight the need for alternative, long-term treatment options that target the root cause of the disorder, making gene therapy a highly relevant area of research.

Gene therapy has emerged as a promising treatment for monogenic diseases, and CAH is an ideal candidate for such approaches due to its well-defined genetic cause. While gene-addition strategies have shown success in improving the biochemical profile in CAH mouse models, a major challenge remains— the high turnover rate of adrenal cortex cells. Therapies relying on AAV vector-mediated episomal expression have been shown to gradually lose their therapeutic effect over time as vector genomes are diluted in the rapidly regenerating adrenal cortex. Consequently, there is a critical need for gene therapies that can provide long-term, stable gene expression in the adrenal cortex.

CRISPR/Cas9 genome-editing technology has revolutionized gene therapy, offering the potential to permanently correct genetic mutations. In the context of CAH, the ability to knock in a functional copy of the 21-hydroxylase gene into the genome of adrenal cortex cells using the CRISPR/Cas9 system could provide a lasting solution by addressing the underlying genetic defect. Genome-editing technology offers the potential for a curative, one-time treatment with durable therapeutic effects. The restoration of normal steroidogenesis may significantly reduce or even eliminate the need for lifelong hormone replacement therapy, along with the associated long-term complications.

This study addresses the unmet clinical needs of CAH by targeting the limitations of hormone replacement therapy and advancing CRISPR/Cas9-based genome-editing therapies. The research contributes to the development of long-term, curative treatments for CAH and potentially other genetic diseases.

Scientific novelty

All the results presented in this thesis are original. To our knowledge, this proof-of-concept study

represents the first application of CRISPR/Cas9-mediated genome editing for the treatment of CAH due to 21-OHD. This novel approach addresses the underlying genetic cause of CAH, offering a potential long-term solution through targeted, site-specific knock-in of the Cyp21a1 gene in adrenal cells. It is a pioneering effort, as previous gene therapies for CAH primarily focused on gene addition rather than genome-editing therapies. Additionally, this study introduces, for the first time, the synthetic AAV-DJ serotype for efficient gene delivery to the adrenal glands. The comprehensive evaluation of AAV vector biodistribution across multiple tissues contributes to the safety profile of AAV-mediated therapies. The study also addresses a methodological challenge by conducting the first systematic investigation of autofluorescence in adrenal tissue sections and by proposing several solutions to improve data quality in fluorescent imaging studies. Importantly, this study goes beyond short-term studies by providing a comprehensive analysis of long-term therapeutic outcomes, including the sustainability of vector genomes and gene expression, steroid hormone production, body weight, and survival rates in Cyp21a1-/- mice.

Theoretical and Practical Significance

This study significantly advances the theoretical understanding of genome-editing therapies, particularly in the context of monogenic disorders like CAH. It enriches the existing literature by demonstrating the use of CRISPR/Cas9 not only as a tool for gene knock-out but also as a mechanism for gene knock-in, offering stable therapeutic effects through the integration of functional genes at specific loci. The research provides valuable insights into the efficiency of DNA repair mechanisms, specifically homology-directed repair (HDR) and non-homologous end joining (NHEJ) during genome editing. Emphasizing that NHEJ is more efficient than HDR in mediating gene knock-in is important for optimizing genome-editing strategies. This knowledge enhances a broader understanding of how DNA repair pathways can be applied therapeutically.

The study expands knowledge of AAV vectors by investigating AAV serotype tropism to the adrenal glands. The finding that synthetic AAV-DJ exhibits strong adrenal cortex and liver tropism informs vector engineering to improve gene delivery, supporting further research to optimize vector tropism. Additionally, systemic IV AAV administration yields higher adrenal gene expression than local injections, which are constrained by injection volume and do not enhance AAV transduction specificity. These findings suggest that vascularization and blood supply are key factors influencing AAV biodistribution.

The study's practical significance lies in its potential to drive the development of new therapies for CAH. Demonstrating that AAV-based CRISPR/Cas9-based genome-editing therapy restores steroid

hormone production in a CAH mouse model, the research paves the way for more effective, long-lasting treatments that could reduce or eliminate lifelong hormone replacement therapy, improving patient quality of life. The findings extend beyond CAH to other adrenal and monogenic disorders.

Furthermore, the research provides valuable data on the long-term efficacy of gene therapies by monitoring therapeutic outcomes over an extended period. These findings are highly relevant for clinical translation, where maintaining long-term gene expression poses a significant challenge. The sustained therapeutic effects demonstrated by AAV-based CRISPR/Cas9 genome editing over time support the clinical viability of these approaches. The study also offers protocols and tools for future gene therapy research. Methods for genotyping, evaluating vector genome copy numbers, assessing mRNA expression in tissues, and autofluorescence quenching in adrenal tissues offer valuable resources for other researchers engaged in both preclinical and clinical studies.

Author's contribution

The results presented in this thesis were obtained from the scientific research conducted by the applicant as a PhD student at the Moscow Institute of Physics and Technology (MIPT, Phystech). The applicant conducted the research under the supervision of Dr. Pavel Volchkov, with scientific and medical consultation provided by Dr. Maria Vorontsova. Conducting experiments, obtaining results, conducting data analysis, and writing the thesis were primarily undertaken by the applicant or with his direct participation. Experimental design and preparation of manuscripts for publication were completed by the applicant or in active collaboration with Dr. Olga Glazova. Flow cytometry analysis was conducted with assistance from Ekaterina Antonova. Animal procedures were carried out at the Faculty of Medicine, M.V. Lomonosov Moscow State University, with assistance from Liudmila Shevkova and Alena Shilova, under the guidance of Dr. Vladimir Popov. Confocal microscopy imaging was performed at the Endocrinology Research Centre and the Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies. Steroid hormone analyses were performed at the Endocrinology Research Centre and Arkhimed clinic (Moscow).

Statements to be defended

1. Systemic administration of AAV vectors results in efficient delivery and strong gene expression in adrenal cortex cells. The synthetic AAV-DJ serotype demonstrates strong tropism for both the adrenal glands and liver, supporting its suitability for developing gene therapy strategies for adrenal genetic disorders.

2. The non-homologous end joining repair pathway enables efficient site-specific knock-in of the Cyp21a1 expression cassette at CRISPR/Cas9-induced double-strand breaks in vitro and in vivo, confirming its applicability for stable genome editing in regenerative tissues.

3. This proof-of-concept study presents the first application of CRISPR/Cas9-mediated genome editing for the treatment of 21-OHD in an animal model. AAV-based CRISPR/Cas9-mediated knock-in of the Cyp21a1 expression cassette partially corrects steroidogenesis and ensures long-term survival in Cyp21a1-/- mice, demonstrating the therapeutic potential of genome-editing therapy for CAH.

Approbation of the thesis

The main results of the work were presented at the following scientific conferences and seminars:

1. The 64th All-Russian Scientific Conference of the Moscow Institute of Physics and Technology, Dolgoprudny, Russia, 2021.

2. Moscow Workshop on the Prospects of Gene and Cell Therapy, Dolgoprudny, Russia, 2022.

3. Scientific seminar "Development of gene therapy for a mouse model of congenital adrenal hyperplasia using genome editing techniques", Life Sciences Research Center of MIPT, Dolgoprudny, Russia, 2023.

4. IV Young Scientists Congress, Sochi, Russia, 2024.

Publications

1. Sakr, N., Glazova, O., Shevkova, L., Onyanov, N., Kaziakhmedova, S., Shilova, A., Vorontsova, M.V., Volchkov, P. Characterizing and Quenching Autofluorescence in Fixed Mouse Adrenal Cortex Tissue. International Journal of Molecular Sciences 2023, 24(4):3432.

2. Glazova O., Bastrich A., Deviatkin A., Onyanov N., Kaziakhmedova S., Shevkova L., Sakr N., Petrova D., Vorontsova M., Volchkov P. Models of Congenital Adrenal Hyperplasia for Gene Therapies Testing. International Journal of Molecular Sciences 2023, 24(6):5365.

3. Glazova O., Vorontsova M., Sakr N., Shevkova L., Onyanov N., Kaziakhmedova S., Volchkov P.Y. Gene and cell therapy of adrenal pathology: achievements and prospects. Problemy Endokrinologii 2021, 67(6):80-89

4. Patent No. 2812468 C1. Expression vectors based on adeno-associated virus / Glazova O.V., Shevkova L.V., Sakr N., Vorontsova M.V., Volchkov P.Y.; applicant and rights holder OOO "Geneticheskie tekhnologii". - No. 2023120008; filed 29.07.2023; published 30.01.2024, Bulletin No. 4.

5. Patent No. 2812469 C1. Combination of vectors for therapy of congenital dysfunction of adrenal cortex / Glazova O.V., Shevkova L.V., Sakr N., Vorontsova M.V., Volchkov P.Y.; applicant and rights holder OOO "Geneticheskie tekhnologii". - No. 2023120009; filed 29.07.2023; published 30.01.2024, Bulletin No. 4.

Structure and size of the work

The thesis consists of the following sections: Abstract, Introduction, Literature Review, Materials and Methods, Results, Discussion, Conclusions, Main Results and Outlook, and References. The thesis includes 178 pages and 55 figures. The reference list contains 247 references.

Заключение диссертации по теме «Другие cпециальности», Сакр Навар

Conclusion

This proof-of-concept study investigated CRISPR/Cas9-mediated knock-in of a functional Cyp21a1 gene at the Rosa26 safe-harbor locus as a therapeutic strategy for CAH in a 21-OHD mouse model. This AAV-based genome-editing strategy is designed to overcome dilution and loss of AAV-mediated expression caused by the adrenal cortex's high regenerative capacity. Systemic AAV-mediated delivery of the CRISPR/Cas9 system and the knock-in donor cassette enabled a stable integration of the Cyp21a1 expression cassette in the adrenal glands and liver tissues via non-homologous end joining, supporting its potential for genome-editing therapy in regenerative tissues.

The AAV-based genome-editing therapy achieved a durable benefit in Cyp21a1-- mice. It maintained stable persistence of the Cyp21a1 expression cassette in adrenal tissue, supported long-term restoration of 11-deoxycorticosterone synthesis, and ensured 100% survival over 28 weeks. These findings support genome editing as a viable strategy for sustained functional correction in CAH.

Despite the promising results of this study, several challenges remain. The strong tropism of wildtype AAV and AAV-DJ serotypes for the liver tissue raises potential concerns for clinical applications, particularly regarding off-target effects and immune responses. Enhancing adrenal-specific expression and editing using tissue-specific promoters and modified AAV capsids will be crucial for improving safety and therapeutic outcomes. Additionally, further investigation is needed to address knock-in efficiency, off-target genome editing, and the long-term effects of CRISPR/Cas9 activity.

This thesis provides valuable insights into the development of genome-editing therapy for CAH as a promising strategy to overcome the limitations of gene-addition therapy. Future studies should focus on refining vector design, enhancing adrenal targeting, and improving editing efficiency while ensuring safety for clinical translation. Overall, the findings presented here establish a strong foundation for advancing CRISPR/Cas-based therapies toward clinical application, paving the way for next-generation treatments with curative potential for CAH and other monogenic endocrine disorders.

Main results and the outlook

1. AAV expression cassettes were constructed and validated. The assembled AAV vectors enabled Cyp21a1 and Cas9 expression in vitro and in vivo.

2. Systemic AAV administration resulted in high vector genome copy numbers and strong transgene expression in mouse adrenal glands.

3. The synthetic AAV-DJ serotype demonstrated strong tropism for the mouse adrenal cortex and liver following systemic administration.

4. CRISPR/Cas9-mediated knock-in via non-homologous end joining enabled stable site-specific integration of the Cyp21a1 expression cassette in mouse adrenal and hepatic cells.

5. Genome-editing therapy with AAV-Cyp21 and AAV-Cas9 resulted in sustained restoration of 11-deoxycorticosterone synthesis, prolonged body-weight improvement, and 100% survival in Cyp21a1-/- mice over 28 weeks post-treatment.

Список литературы диссертационного исследования кандидат наук Сакр Навар, 2025 год

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