CDKL5 deficiency disorder (CDD) is a rare X-linked neurodevelopmental disorder characterized by early-onset, drug-resistant epilepsy, severe cognitive impairment, motor dysfunction, and autistic features. Despite advances in the understanding of its molecular and cellular basis, current treatments remain largely symptomatic, and no disease-modifying treatments are available. One of the major challenges in developing effective therapies for CDD lies in achieving efficient, widespread, and sustained delivery of therapeutic molecules to the central nervous system (CNS), particularly to brain regions that are poorly accessible using conventional viral delivery. Among emerging strategies, gene therapy has shown considerable promise in preclinical models. While adeno-associated viral (AAV) vectors have shown promising results, their efficacy is often limited by restricted distribution within the brain. To address this, a cross-correction strategy employing a secretable and cell-penetrating CDKL5 fusion protein (IgK-TATk-CDKL5) has been developed to improve neuroanatomical and behavioral deficits in Cdkl5 knockout (KO) mouse models. However, the localized nature of AAV injections often results in suboptimal protein delivery to distal critical brain regions. Hematopoietic stem cell (HSC)–based gene therapy represents a complementary strategy that may overcome these limitations. Following ex vivo genetic modification and transplantation, HSCs can engraft long-term and differentiate into progeny, including microglia-like cells that populate the CNS. These donor-derived cells can act as local sources of therapeutic molecules, enabling sustained and widespread protein distribution within the brain. This approach has already demonstrated clinical and preclinical success in other neurological disorders, supporting its potential application in diseases requiring broad CNS correction. In this work, a combined gene and cell therapy strategy was explored to enhance CDKL5 delivery in the CNS. A lentiviral vector encoding a secretable and cell penetrating CDKL5 fusion protein (IgK-TAT-hCDKL5.CO*HA*) was generated. The vector was characterized for transduction efficiency and functional performance. Lineage-negative (Lin⁻) hematopoietic stem cells were isolated from donor mice, transduced ex vivo and subsequently transplanted into Cdkl5 knockout (CDKL5 -/Y and CDKL5 -/-) recipients. Molecular analyses were performed to assess the presence and expression of the CDKL5 transgene following transplantation, as well as the extent of donor-derived cell contribution and engraftment within the recipient brain. Particular attention was given to quantifying cellular engraftment and microglial reconstitution to evaluate the efficiency of this approach and determine whether it could improve local delivery compared to conventional viral-based strategies. Overall, this work demonstrates the potential of HSC-based gene therapy as a platform to achieve sustained and widespread delivery of therapeutic proteins in the CNS. By combining the advantages of cross-correction mechanisms with the intrinsic properties of hematopoietic stem cells, this approach may represent a promising strategy to overcome current limitations in the treatment of CDKL5 deficiency disorder.
Il disturbo da deficit di CDKL5 (CDKL5 Deficiency Disorder, CDD) è una patologia del neurosviluppo legata al cromosoma X, caratterizzata da epilessia farmacoresistente a esordio precoce, compromissione cognitiva, disfunzioni motorie e manifestazioni riconducibili allo spettro autistico. Nonostante i progressi nella comprensione delle basi molecolari e cellulari della malattia, le attuali opzioni terapeutiche rimangono prevalentemente sintomatiche e non sono ancora disponibili trattamenti in grado di modificare il decorso della patologia. Una delle principali sfide nello sviluppo di terapie efficaci per il CDD consiste nel garantire una distribuzione efficiente e duratura delle molecole terapeutiche all’interno del sistema nervoso centrale (SNC), in particolare nelle regioni cerebrali difficilmente raggiungibili mediante i tradizionali approcci di veicolazione virale. Tra le strategie emergenti, la terapia genica ha mostrato risultati promettenti in modelli preclinici. Sebbene i vettori adeno-associati (AAV) abbiano evidenziato un potenziale terapeutico, la loro efficacia risulta spesso limitata dalla ridotta diffusione all’interno del parenchima cerebrale. Per superare tale limitazione, è stata sviluppata una strategia di cross-correction basata sull’impiego di una proteina di fusione CDKL5 secreta e capace di penetrare nelle cellule (IgK-TATk-CDKL5), che ha dimostrato di migliorare alterazioni neuroanatomiche e comportamentali nei modelli murini knockout (KO) per Cdkl5. Tuttavia, la natura localizzata delle iniezioni di AAV può determinare una distribuzione subottimale della proteina nelle regioni cerebrali distali di rilevanza funzionale. La terapia genica basata sulle cellule staminali ematopoietiche (Hematopoietic Stem Cells, HSC) rappresenta una strategia complementare in grado di superare tali limitazioni. In seguito alla modificazione genetica ex vivo e al successivo trapianto, le HSC possono attecchire stabilmente e differenziarsi in diverse popolazioni, comprese cellule simil-microgliali capaci di colonizzare il SNC. Queste cellule derivate dal donatore possono fungere da sorgenti locali di molecole terapeutiche, consentendo una distribuzione prolungata e diffusa della proteina all’interno del cervello. Tale approccio ha già dimostrato successo in ambito preclinico e clinico per altre patologie neurologiche, nelle malattie che richiedono una correzione estesa a livello del SNC. Nel presente lavoro è stata esplorata una strategia combinata di terapia cellulare e genica finalizzata a migliorare la distribuzione di CDKL5 nel SNC. È stato generato un vettore lentivirale codificante per una proteina di fusione CDKL5 secreta ed in grado di penetrare nelle cellule (IgK-TAT hCDKL5.CO.HA). Il vettore è stato caratterizzato in termini di efficienza di trasduzione e funzionalità biologica. Cellule staminali ematopoietiche sono state isolate da topi donatori, trasdotte ex vivo e successivamente trapiantate in modelli murini knockout per Cdkl5 (Cdkl5 -/Y e Cdkl5 -/-). Sono state condotte analisi molecolari per valutare la presenza e l’espressione del transgene CDKL5 in seguito al trapianto, nonché il contributo e l’attecchimento delle cellule derivate dal donatore nel cervello dei riceventi. È stata inoltre valutata la quantificazione dell’attecchimento cellulare e della ricostituzione della popolazione microgliale, confermando l’efficienza di questo approccio nel modello murino di CDD. Complessivamente, questo studio dimostra il potenziale della terapia genica basata sulle cellule staminali ematopoietiche come piattaforma per ottenere una distribuzione prolungata e diffusa di proteine terapeutiche nel sistema nervoso centrale. Combinando i meccanismi di cross-correction con le proprietà delle cellule staminali ematopoietiche, questo approccio potrebbe rappresentare una strategia promettente per superare le attuali limitazioni terapeutiche nel trattamento del disturbo da deficit di CDKL5.
Terapia genica delle cellule staminali ematopoietiche come approccio terapeutico per il disturbo da deficit di CDKL5, valutata in un modello murino della malattia
KATSANOU, LAMPRINI
2025/2026
Abstract
CDKL5 deficiency disorder (CDD) is a rare X-linked neurodevelopmental disorder characterized by early-onset, drug-resistant epilepsy, severe cognitive impairment, motor dysfunction, and autistic features. Despite advances in the understanding of its molecular and cellular basis, current treatments remain largely symptomatic, and no disease-modifying treatments are available. One of the major challenges in developing effective therapies for CDD lies in achieving efficient, widespread, and sustained delivery of therapeutic molecules to the central nervous system (CNS), particularly to brain regions that are poorly accessible using conventional viral delivery. Among emerging strategies, gene therapy has shown considerable promise in preclinical models. While adeno-associated viral (AAV) vectors have shown promising results, their efficacy is often limited by restricted distribution within the brain. To address this, a cross-correction strategy employing a secretable and cell-penetrating CDKL5 fusion protein (IgK-TATk-CDKL5) has been developed to improve neuroanatomical and behavioral deficits in Cdkl5 knockout (KO) mouse models. However, the localized nature of AAV injections often results in suboptimal protein delivery to distal critical brain regions. Hematopoietic stem cell (HSC)–based gene therapy represents a complementary strategy that may overcome these limitations. Following ex vivo genetic modification and transplantation, HSCs can engraft long-term and differentiate into progeny, including microglia-like cells that populate the CNS. These donor-derived cells can act as local sources of therapeutic molecules, enabling sustained and widespread protein distribution within the brain. This approach has already demonstrated clinical and preclinical success in other neurological disorders, supporting its potential application in diseases requiring broad CNS correction. In this work, a combined gene and cell therapy strategy was explored to enhance CDKL5 delivery in the CNS. A lentiviral vector encoding a secretable and cell penetrating CDKL5 fusion protein (IgK-TAT-hCDKL5.CO*HA*) was generated. The vector was characterized for transduction efficiency and functional performance. Lineage-negative (Lin⁻) hematopoietic stem cells were isolated from donor mice, transduced ex vivo and subsequently transplanted into Cdkl5 knockout (CDKL5 -/Y and CDKL5 -/-) recipients. Molecular analyses were performed to assess the presence and expression of the CDKL5 transgene following transplantation, as well as the extent of donor-derived cell contribution and engraftment within the recipient brain. Particular attention was given to quantifying cellular engraftment and microglial reconstitution to evaluate the efficiency of this approach and determine whether it could improve local delivery compared to conventional viral-based strategies. Overall, this work demonstrates the potential of HSC-based gene therapy as a platform to achieve sustained and widespread delivery of therapeutic proteins in the CNS. By combining the advantages of cross-correction mechanisms with the intrinsic properties of hematopoietic stem cells, this approach may represent a promising strategy to overcome current limitations in the treatment of CDKL5 deficiency disorder.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/36092