Preclinical Milestones in MECP2 Gene Transfer for Treating Rett Syndrome.

BACKGROUND: Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2). After gene transfer in mice, exogenous MeCP2 expression must be regulated to avoid dose-dependent toxicity. SUMMARY: The preclinical gene therapy literature for treating RTT illustrates a duly diligent progression that begins with proof-of-concept studies and advances toward the development of safer, regulated MECP2 viral genome designs. This design progression was partly achieved through international collaborative studies. In 2023, clinicians administered investigational gene therapies for RTT to patients a decade after the first preclinical gene therapy publications for RTT (clinical trial numbers NCT05606614 and NCT05898620). As clinicians take on a more prominent role in MECP2 gene therapy research, preclinical researchers may continue to test more nuanced hypotheses regarding the safety, efficacy, and mechanism of MECP2 gene transfer. KEY MESSAGE: This review summarizes the history of preclinical MECP2 gene transfer for treating RTT and acknowledges major contributions among colleagues in the field. The first clinical injections are a shared milestone.

Engineered microRNA-based regulatory element permits safe high-dose miniMECP2 gene therapy in Rett mice.

MECP2 gene transfer has been shown to extend the survival of Mecp2-/y knockout mice modelling Rett syndrome, an X-linked neurodevelopmental disorder. However, controlling deleterious overexpression of MECP2 remains the critical unmet obstacle towards a safe and effective gene therapy approach for Rett syndrome. A recently developed truncated miniMECP2 gene has also been shown to be therapeutic after AAV9-mediated gene transfer in knockout neonates. We show that AAV9/miniMECP2 has a similar dose-dependent toxicity profile to that of a published second-generation AAV9/MECP2 vector after treatment in adolescent mice. To overcome that toxicity, we developed a risk-driven viral genome design strategy rooted in high-throughput profiling and genome mining to rationally develop a compact, synthetic microRNA target panel (miR-responsive auto-regulatory element, 'miRARE') to minimize the possibility of miniMECP2 transgene overexpression in the context of Rett syndrome gene therapy. The goal of miRARE is to have a built-in inhibitory element responsive to MECP2 overexpression. The data provided herein show that insertion of miRARE into the miniMECP2 gene expression cassette greatly improved the safety of miniMECP2 gene transfer without compromising efficacy. Importantly, this built-in regulation system does not require any additional exogenous drug application, and no miRNAs are expressed from the transgene cassette. Although broad applications of miRARE have yet to be determined, the design of miRARE suggests a potential use in gene therapy approaches for other dose-sensitive genes.

The Efficacy of a Human-Ready miniMECP2 Gene Therapy in a Pre-Clinical Model of Rett Syndrome.

Inactivating mutations and the duplication of methyl-CpG binding protein 2 (MeCP2), respectively, mediate Rett syndrome (RTT) and MECP2 duplication syndrome. These disorders underscore the conceptual dose-dependent risk posed by MECP2 gene therapy for mosaic RTT patients. Recently, a miRNA-Responsive Autoregulatory Element (miRARE) mitigated the dose-dependent toxicity posed by self-complementary adeno-associated viral vector serotype 9 (AAV9) miniMECP2 gene therapy (scAAV9/miniMECP2-myc) in mice. Here, we report an efficacy assessment for the human-ready version of this regulated gene therapy (TSHA-102) in male Mecp2-/y knockout (KO) mice after intracerebroventricular (ICV) administration at postnatal day 2 (P2) and after intrathecal (IT) administration at P7, P14 (±immunosuppression), and P28 (±immunosuppression). We also report qPCR studies on KO mice treated at P7-P35; protein analyses in KO mice treated at P38; and a survival safety study in female adult Mecp2-/+ mice. In KO mice, TSHA-102 improved respiration, weight, and survival across multiple doses and treatment ages. TSHA-102 significantly improved the front average stance and swing times relative to the front average stride time after P14 administration of the highest dose for that treatment age. Viral genomic DNA and miniMECP2 mRNA were present in the CNS. MiniMeCP2 protein expression was higher in the KO spinal cord compared to the brain. In female mice, TSHA-102 permitted survivals that were similar to those of vehicle-treated controls. In all, these pivotal data helped to support the regulatory approval to initiate a clinical trial for TSHA-102 in RTT patients (clinical trial identifier number NCT05606614).

Recent endeavors in MECP2 gene transfer for gene therapy of Rett syndrome.

Rett Syndrome (RTT) is an X chromosome-linked neurodevelopmental disorder caused by inactivating mutations in the transcription regulator methyl CpG-binding protein 2 (MeCP2). Multiple studies have independently explored the therapeutic potential of adeno-associated viral (AAV) vector-mediated MECP2 gene transfer in mouse models of RTT. Historically, the primary risk anticipated for viral vector-mediated MECP2 gene transfer in vivo has been toxicity caused by supraphysiological expression of exogenous MeCP2. Despite the anticipated risk, early studies examining AAV/MECP2 in vivo have, as a whole, supported a generally optimistic assessment of MECP2 gene therapy. More recently, toxicity assessments have identified dose-dependent side effects of AAV9/MECP2 delivered directly to the cerebrospinal fluid (CSF). Ultimately, accurate monitoring and reporting of these side effects will help ensure the development of safe AAV/MECP2 treatment paradigms as researchers explore strategies to improve widespread but properly regulated MECP2 gene transfer in the central nervous system (CNS). Importantly, despite some variability in apparent safety and efficacy, all MECP2 gene therapy studies have been united by a single feat: published treatment paradigms have extended the survival of RTT mice, regardless of injection route, treatment age, or viral genome design. With the possibility of a translatable gene therapy treatment for RTT emerging, a comprehensive overview of the preclinical MECP2 gene therapy studies published thus far is warranted. This review highlights the main findings of these publications and discusses future directions.

Improved MECP2 Gene Therapy Extends the Survival of MeCP2-Null Mice without Apparent Toxicity after Intracisternal Delivery.

Intravenous administration of adeno-associated virus serotype 9 (AAV9)/hMECP2 has been shown to extend the lifespan of Mecp2-/y mice, but this delivery route induces liver toxicity in wild-type (WT) mice. To reduce peripheral transgene expression, we explored the safety and efficacy of AAV9/hMECP2 injected into the cisterna magna (ICM). AAV9/hMECP2 (1 × 1012 viral genomes [vg]; ICM) extended Mecp2-/y survival but aggravated hindlimb clasping and abnormal gait phenotypes. In WT mice, 1 × 1012 vg of AAV9/hMECP2 induced clasping and abnormal gait. A lower dose mitigated these adverse phenotypes but failed to extend survival of Mecp2-/y mice. Thus, ICM delivery of this vector is impractical as a treatment for Rett syndrome (RTT). To improve the safety of MeCP2 gene therapy, the gene expression cassette was modified to include more endogenous regulatory elements believed to modulate MeCP2 expression in vivo. In Mecp2-/y mice, ICM injection of the modified vector extended lifespan and was well tolerated by the liver but did not rescue RTT behavioral phenotypes. In WT mice, these same doses of the modified vector had no adverse effects on survival or neurological phenotypes. In summary, we identified limitations of the original vector and demonstrated that an improved vector design extends Mecp2-/y survival, without apparent toxicity.

The Role of AMPK in Drosophila melanogaster.

In the fruit fly, Drosophila melanogaster, mono-allelic expression of AMPK-α, -ß, and -γ yields a single heterotrimeric energy sensor that regulates cellular and whole-body energetic homeostasis. The genetic simplicity of Drosophila, with only a single gene for each subunit, makes the fruit fly an appealing organism for elucidating the effects of AMPK mutations on signaling pathways and phenotypes. In addition, Drosophila presents researchers with an opportunity to use straightforward genetic approaches to elucidate metabolic signaling pathways that contain a level of complexity similar to that observed in mammalian pathways. Just as in mammals, however, the regulatory realm of AMPK function extends beyond metabolic rates and lipid metabolism. Indeed, experiments using Drosophila have shown that AMPK may exert protective effects with regard to life span and neurodegeneration. This chapter addresses a few of the research areas in which Drosophila has been used to elucidate the physiological functions of AMPK. In doing so, this chapter provides a primer for basic Drosophila nomenclature, thereby eliminating a communication barrier that persists for AMPK researchers trained in mammalian genetics.

Past strategies and future directions for identifying AMP-activated protein kinase (AMPK) modulators.

AMP-activated protein kinase (AMPK) is a promising therapeutic target for cancer, type II diabetes, and other illnesses characterized by abnormal energy utilization. During the last decade, numerous labs have published a range of methods for identifying novel AMPK modulators. The current understanding of AMPK structure and regulation, however, has propelled a paradigm shift in which many researchers now consider ADP to be an additional regulatory nucleotide of AMPK. How can the AMPK community apply this new understanding of AMPK signaling to translational research? Recent insights into AMPK structure, regulation, and holoenzyme-sensitive signaling may provide the hindsight needed to clearly evaluate the strengths and weaknesses of past AMPK drug discovery efforts. Improving future strategies for AMPK drug discovery will require pairing the current understanding of AMPK signaling with improved experimental designs.

A High Throughput Assay for Discovery of Small Molecules that Bind AMP-activated Protein Kinase (AMPK).

AMPK is a conserved heterotrimeric serine-threonine kinase that regulates anabolic and catabolic pathways in eukaryotes. Its central role in cellular and whole body metabolism makes AMPK a commonly proposed therapeutic target for illnesses characterized by abnormal energy regulation, including cancer and diabetes. Many AMPK modulators, however, produce AMPK-independent effects. To identify drugs that modulate AMPK activity independent of the canonical ATP-binding pocket found throughout the kinome, we designed a robust fluorescence-based high throughput screening assay biased toward the identification of molecules that bind the regulatory region of AMPK through displacement of MANT-ADP, a fluorescent ADP analog. Automated pin tools were used to rapidly transfer small molecules to a low volume assay mixture on 384-well plates. Prior to assay validation, we completed a full assay optimization to maximize the signal-to-background and reduce variability for robust detection of small molecules displacing MANT-ADP. After validation, we screened 13,120 molecules and identified 3 positive hits that dose-dependently inhibited the protein-bound signal of MANT-ADP in the presence of both full-length AMPK and the truncated "regulatory fragment" of AMPK, which is missing the kinase active site. The average Z'-factor for the screen was 0.55 and the compound confirmation rate was 60%. Thus, this fluorescence-based assay may be paired with in vitro kinase assays and cell-based assays to help identify molecules that selectively regulate AMPK with fewer off-target effects on other kinases.

Effects of a stressor and corticotrophin releasing factor on ethanol deprivation-induced ethanol intake and anxiety-like behavior in alcohol-preferring P rats.

RATIONALE: Stress may elevate ethanol drinking and anxiety associated with ethanol drinking. Studies to identify relevant neurobiological substrates are needed. OBJECTIVE: To assess roles of brain regions in corticotrophin releasing factor (CRF) effects on stressor-enhanced, ethanol deprivation-induced drinking and anxiety-like behavior. METHODS: Ethanol-preferring rats (P rats) were exposed to three cycles of a two-bottle choice paradigm with two 2-day deprivation periods that included 1 h exposure to a restraint stressor. To assess the role of CRF and to identify relevant brain regions, a CRF-1 receptor antagonist (SSR125543; 10 ug) was injected into the nucleus accumbens (NAC), amygdala (Amyg), or dorsal raphe nucleus (DRN) prior to exposure to the restraint stressor. In a second study, CRF (0.5 ug) was injected into one of these regions, or the ventral tegmental area (VTA), or paraventricular nucleus of the hypothalamus (PVN). RESULTS: Applying the restraint stressor during deprivation increased voluntary intake and sensitized anxiety-like behavior. Antagonist injection into the NAC prevented increased drinking without affecting anxiety-like behavior, whereas injection into the Amyg or DRN prevented the anxiety-like behavior without affecting drinking. To confirm CRF actions in the stressor effect, CRF was injected into selected brain regions. NAC injections (but not the VTA, Amyg, DRN, or PVN) facilitated drinking but did not change anxiety-like behavior. Injections into the DRN or Amyg (but not PVN or VTA) enhanced anxiety-like behavior. CONCLUSIONS: Results emphasize that a restraint stressor elevates ethanol intake and sensitizes ethanol deprivation-induced anxiety-like behavior through CRF1 receptors in the NAC and Amyg/DRN, respectively.