Use of a quantitative partial reprogramming-based nucleic acid composition in the preparation of a medicament for preventing and treating Alzheimer's disease

Abstract

This invention relates to the field of biomedical technology, and more particularly to the application of a quantitatively partially reprogrammed nucleic acid composition in the preparation of drugs for the prevention and treatment of Alzheimer's disease. The invention provides an application of a quantitatively partially reprogrammed nucleic acid composition in the preparation of drugs for the prevention and treatment of Alzheimer's disease, wherein the composition comprises nucleic acid constructs encoding Oct4, Sox2, Klf4, Glis1, and Lin28 proteins. This invention constitutes a complete closed-loop system from intervention, monitoring, to regulation, encompassing drug combination, inducible neural tropism delivery vectors, efficacy monitoring methods based on peripheral methylation clocks, and a clearly defined safe therapeutic window. This system is not only suitable for intracranial injection but also compatible with non-invasive methods such as intranasal targeted delivery, providing a practical and feasible translational pathway for the precise prevention and treatment of Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a nucleic acid composition based on quantitative partial reprogramming in the preparation of drugs for the prevention and treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is the leading cause of dementia, and its core pathological features include neurofibrillary tangles formed by β-amyloid (Aβ) deposition and Tau protein hyperphosphorylation. Current therapies can only provide limited symptom relief and cannot reverse the disease progression. Recent studies have shown widespread epigenetic dysregulations (especially DNA methylation pattern disruptions) in the AD brain, preceding obvious pathological changes, which are considered key upstream events driving neuronal dysfunction and neuroinflammation.

[0003] Partial reprogramming techniques, through the transient expression of reprogramming factors, have been shown to reset the epigenetic age of cells, restoring their function and vitality. However, applying this technology to the treatment of Alzheimer's disease faces unique challenges: Complexity and safety of brain tissue: Precise targeting of vulnerable neurons is required while avoiding interference with normal neural circuits or inducing abnormal proliferation. Bottlenecks in efficacy assessment: A lack of objective, quantifiable biomarkers reflecting the fundamental epigenetic process of the disease. Clinically, while strategies directly targeting Aβ or Tau have shown some effectiveness, they often have limitations. Unknown therapeutic window: The degree to which epigenetic reprogramming can achieve a balance between maximizing neural repair and minimizing risk in the brain is currently completely unknown.

[0004] Therefore, there is an urgent need in this field for a new AD treatment strategy that can directly intervene in the core epigenetic drivers of the disease, has objective quantitative efficacy monitoring methods, and has clear intracranial safety boundaries. Summary of the Invention

[0005] The purpose of this invention is to provide an application of a nucleic acid composition based on quantitative partial reprogramming in the preparation of drugs for the prevention and treatment of Alzheimer's disease.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the use of a quantitatively partially reprogrammed nucleic acid composition in the preparation of drugs for the prevention and treatment of Alzheimer's disease, the composition comprising nucleic acid constructs encoding Oct4, Sox2, Klf4, Glis1 and Lin28 proteins.

[0007] Preferably, the nucleic acid construct does not encode the c-Myc protein.

[0008] Preferably, the nucleic acid construct is carried in a neural tropism delivery vector.

[0009] Preferably, the neural tropism delivery vector is a recombinant adeno-associated virus vector, a nasal targeted delivery system, or a combination thereof.

[0010] Preferably, the nucleic acid construct contains an inducible gene expression regulatory element.

[0011] Preferably, the inducible gene expression regulatory element is a tetracycline-inducible expression system.

[0012] Preferably, the nucleic acid construct comprises a combination of the following three recombinant adeno-associated virus vectors: (1) A first recombinant adeno-associated virus vector containing nucleic acid sequences encoding Oct4, Sox2 and Klf4 proteins that are controlled by an inducible promoter; (2) A second recombinant adeno-associated virus vector, comprising the nucleic acid sequences encoding Glis1 and Lin28 proteins controlled by the inducible promoter; and (3) The third recombinant adeno-associated virus vector contains a nucleic acid sequence encoding a reverse tetracycline transactivator linked by a constitutive promoter.

[0013] Preferably, in the first recombinant adeno-associated virus vector, the nucleic acid sequences encoding Oct4, Sox2, and Klf4 proteins are tandemly linked by self-cleaving peptide sequences; in the second recombinant adeno-associated virus vector, the nucleic acid sequences encoding Glis1 and Lin28 proteins are tandemly linked by self-cleaving peptide sequences.

[0014] Preferably, the inducible promoter is the TRE3G promoter, and the constitutive promoter is the human ubiquitin C promoter; the reverse tetracycline trans-activator is a modified rtTAX, which is a variant obtained by introducing three point mutations, T45A, S95N and L138V, on the rtTA2s-M2 amino acid sequence.

[0015] The present invention provides a pharmaceutical composition for the treatment of Alzheimer's disease, comprising the aforementioned nucleic acid construct and a pharmaceutically acceptable carrier or excipient.

[0016] In this invention, the Epigenetic Age Acceleration (EAA) is defined as the difference between an individual's predicted epigenetic age and their actual chronological age. It is a quantitative indicator reflecting the degree to which the rate of cellular biological aging deviates from the baseline of the normal population. At the biological level, the formation of EAA stems from the systematic drift of the DNA methylome during the aging process—the methylation state of specific CpG sites changes with age in a highly regular manner. Pathological factors such as disease, oxidative stress, chronic inflammation, and genotoxic damage can accelerate this drift process, causing an individual's methylome characteristics to be ahead of the normal state corresponding to their chronological age. Therefore, EAA is essentially a molecular imprint of "biological depletion" at the cellular level. Its elevation signifies a measurable decline in the cell's ability to maintain gene expression homeostasis, respond to DNA damage, and perform normal metabolic functions.

[0017] In Alzheimer's disease patients, both brain neurons and circulating peripheral blood mononuclear cells show a significant positive shift in EAA, indicating an "epigenetic accelerated aging" state. This phenomenon can be detected several years before the onset of clinical symptoms, suggesting that epigenetic dysregulation is an upstream event preceding Aβ deposition and Tau hyperphosphorylation, rather than a secondary result of damage. Elevated EAA levels in AD patients are directly causally linked to the following core pathological mechanisms: First, DNA methylome disruption leads to demethylation and activation of neuroinflammation-related genes (such as the regulatory regions of IL-6 and TNF-α), continuously driving excessive microglial activation. Second, abnormally elevated methylation in the promoter regions of neurotrophic factors (such as BDNF) inhibits their transcriptional expression, depriving neurons of survival signal support. Third, epigenetic silencing of synaptic plasticity-related genes (such as SYP and SNAP25) leads to decreased synaptic transmission efficiency, directly corresponding to the clinical manifestations of cognitive decline. Fourth, epigenetic dysregulation of DNA damage repair genes (such as the PARP1-related network) further weakens the neurons' ability to maintain genomic stability under oxidative stress. In summary, EAA is not only a biological reflection of disease severity but also an upstream regulatory node driving the aforementioned pathological cascade.

[0018] The "Reversal Magnitude" (RM) is defined as: RM = [(Baseline EAA - Post-Intervention EAA) / Disease-Related Acceleration Difference] × 100%, where the disease-related acceleration difference is the difference between the average EAA of the AD patient population and the average EAA of healthy age-matched individuals. The biological significance of this indicator lies in its normalization of the intervention-induced epigenetic age change to the reference dimension of "disease burden," thereby transforming absolute methylation changes into a clinically comparable measure of relative efficacy. RM = 0% means that the intervention failed to alter the disease-related epigenetic abnormalities; RM = 100% means that the patient's epigenetic characteristics have completely regressed to the level of healthy age-matched individuals.

[0019] The association between RM and clinical outcomes can be explained at the following three levels: First, at the molecular level, the enhancement of RM corresponds to the recalibration of neuronal gene expression networks. When partial reprogramming intervention reduces EAA to a safe therapeutic window of 60%-75%, the repressive methylation of neuroinflammatory driver genes is restored, and the abnormal hypermethylation in the promoter regions of neurotrophic factor genes such as BDNF and GDNF is effectively removed, restoring their transcriptional activity to near-normal levels, thus providing a molecular basis for the functional repair of synaptic plasticity.

[0020] Second, at the cellular functional level, intervention in the RM 60%-75% range induces neurons and glial cells to enter a state of "functional rejuvenation": mitochondrial oxidative phosphorylation efficiency is improved, autophagy-lysosomal pathway is activated (which helps clear Aβ and phosphorylated Tau protein), and the probability of presynaptic membrane vesicle release is restored. This is manifested in improved neural conduction efficiency, which directly corresponds to the shortened escape latency and improved new object recognition index in the Morris water maze cognitive behavior test.

[0021] Third, at the system level, the quantification of RM provides an operational closed-loop feedback basis for the individualized adjustment of clinical intervention dosage. Due to differences in genetic background, disease stage, and metabolism, the intervention intensity required to achieve the same RM varies significantly among individuals. By dynamically monitoring RM and using 60%-75% as the regulatory target, real-time individualized optimization of the intervention plan can be achieved, avoiding the two extremes of insufficient intervention (RM<60%, limited pathological clearance effect) or excessive intervention (RM>75%, impaired genomic stability, and increased potential tumor risk). This maximizes cognitive benefits while ensuring the long-term safety of the central nervous system.

[0022] Definition and Application of the "Safe Therapeutic Window for Neuroepogenetic Remodeling"

[0023] Based on the above detection system, this invention has found through AD animal model research that there is a key “safe treatment window” of 60%-75% between cognitive function improvement and the magnitude of epigenetic age reversal caused by intervention.

[0024] When the reversal rate is less than 60%, the intervention is insufficient to effectively reverse the AD-related epigenetic "aging lock" and has limited effect on pathological clearance and cognitive improvement.

[0025] When the reversal rate is greater than 75%, the intervention may cause excessive disturbance to the stability of the neuronal genome, potentially leading to safety risks.

[0026] When the reversal rate is strictly controlled within 60%-75%, it can optimally reduce neuroinflammation, promote the clearance of pathological proteins, stabilize synaptic function, and significantly improve cognition.

[0027] Compared with the prior art, the present invention has the following beneficial effects: First, it enables upstream source intervention and non-invasive, precise monitoring of the disease. This invention directly targets the upstream epigenetic disorders (especially DNA methylation disorders) at the core of Alzheimer's disease, resetting the epigenetic age of neurons through quantitative partial reprogramming technology, thus fundamentally slowing disease progression. Simultaneously, it is the first to utilize the DNA methylation clock of peripheral blood mononuclear cells or cerebrospinal fluid samples for dynamic monitoring of treatment efficacy, non-invasively calculating the epigenetic age acceleration value (EAA) and reversal magnitude (RM), greatly improving clinical operability and the traceability of personalized treatment.

[0028] Second, a quantitative regulatory standard unifying safety and efficacy was established. This invention, through studies using an AD animal model, for the first time defined a "safe therapeutic window for neuroepigmoid remodeling." This window, defined as the window where epigenetic age reversal is strictly controlled within 60%-75%, can optimally reduce neuroinflammation, promote the clearance of pathological proteins, and stabilize synaptic function while avoiding excessive disturbance to neuronal genomic stability and potential tumor risks. This discovery transforms complex biological interventions into a precisely regulated quantitative process, providing a closed-loop feedback basis for individualized optimization of clinical dosage.

[0029] Third, it provides a complete closed-loop technology system. This invention comprises a complete closed-loop system from intervention, monitoring to regulation, from drug combinations (a five-factor nucleic acid construct of Oct4, Sox2, Klf4, Glis1, and Lin28 without c-Myc), inducible neural tropism delivery vectors (such as recombinant AAV8 combined with the rtTAX induction system), to efficacy monitoring methods based on peripheral methylation clocks, and finally to a clearly defined safe therapeutic window. This system is not only applicable to intracranial injection but also compatible with non-invasive methods such as intranasal targeted delivery, providing a practical and feasible translational pathway for the precise prevention and treatment of Alzheimer's disease. Attached Figure Description

[0030] Figure 1 The first vector (pAAV8-TRE3G::OSK) map.

[0031] Figure 2 The image shows the spectrum of the second carrier (pAAV8-TRE3G::GL).

[0032] Figure 3 The spectrum is for the third vector (pAAV8-UBC::rtTAX-WPRE3-SV40pA). Detailed Implementation

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1: Method for delivering OSKGL nucleic acid composition to the hippocampus and prefrontal cortex of APP / PS1 transgenic mice using dual AAV8 vectors (AAV8-OSK and AAV8-OGL) combined with the rtTAX induction system.

[0035] 1.1 Design Principles of Dual AAV8 Carriers

[0036] The total length of the full-length coding sequence for the OSKGL five factors (Oct4, Sox2, Klf4, Glis1, Lin28) is approximately 5.8 kb, exceeding the effective packaging capacity limit of a single AAV vector (approximately 4.7 kb). To address this vector capacity limitation, this invention employs a dual AAV8 vector splitting strategy, dividing the five factors into two independent vectors: The first vector (pAAV8-TRE3G::OSK) carries a three-factor polycistronic expression cassette encoding Oct4 (POU5F1, NM_002701, CDS 1065bp), Sox2 (NM_003106, CDS 954bp), and Klf4 (NM_004235, CDS 1518bp). The coding sequences of each factor are sequentially linked by self-cleaved peptide sequences T2A (54bp) and P2A (57bp) (Oct4-T2A-Sox2-P2A-Klf4 arrangement). The total expression cassette length is approximately 3.9 kb. The driving promoter is TRE3G (third-generation tetracycline-responsive element, containing 7 repeat Tet-O sequences, approximately 400bp). The 3' end is configured with a truncated optimized WPRE enhancer (mini-WPRE, approximately 170bp, retaining the gamma and alpha core functional elements) and a compact SV40. The polyA signal (approximately 125 bp, retaining the core AATAAA signal and downstream GU-rich region) results in a total inserted fragment of approximately 4.6 kb, within the effective packaging capacity of AAV8 (approximately 4.7 kb) with a safety margin. Alternatively, a full-length WPRE3 (approximately 250 bp) and standard SV40 polyA (approximately 200 bp) can be used, resulting in a total inserted fragment of approximately 4.8 kb, near the upper limit of AAV8 packaging capacity. Further codon compression of the CDS sequence can then be used to fine-tune the packaging capacity.

[0037] The second vector (pAAV8-TRE3G::GL) carries a two-factor polycistronic expression cassette encoding Glis1 (NM_147193, CDS 1764 bp) and Lin28A (NM_024674, CDS 597 bp), linked by a self-cleaving peptide E2A (57 bp) (Glis1-E2A-Lin28 arrangement). The total cassette length is approximately 2.5 kb. It also employs the TRE3G promoter, WPRE3 enhancer, and SV40 polyA signal, with a total insert length of approximately 3.4 kb. Both vectors explicitly do not contain the nucleic acid sequence encoding the c-Myc protein. The absence of the c-Myc coding sequence is preferred because c-Myc, as a classic proto-oncogene, even when expressed within a transient partial reprogramming intervention window, may still induce excessive neuronal proliferation, dedifferentiation, and a long-term risk of tumor formation, especially in Alzheimer's disease neurons where DNA damage repair capacity is already weakened. The OSKGL five-factor combination excluding c-Myc has been shown to achieve sufficient epigenetic remodeling while maintaining the abnormal cell proliferation detection rate at 0% (see Table 2, treatment window dose group data), providing a fundamental guarantee for the safe control of partial reprogramming in the central nervous system.

[0038] The third carrier (pAAV8-UBC::rtTAX-WPRE3-SV40pA): carries the novel modified reverse tetracycline trans-activator rtTAX designed independently by this invention, driven by the human ubiquitin C (UBC) constitutive promoter, configured with the WPRE3 enhancer and SV40 polyA signal, with a total inserted fragment of about 4.0 kb, which meets the AAV8 packaging capacity requirements.

[0039] The working principle of the above three-vector system is as follows: Under normal feeding conditions, without doxycycline (Dox), rtTAX is in an inactive state, the TRE3G promoter is closed or in a state of very low leakage, and there is no basal expression of OSK and OGL factors. Only during the preset intervention window period, doxycycline (0.2 mg / mL) is administered via drinking water or gavage. rtTAX undergoes a conformational change due to Dox binding, gaining a high affinity for TRE3G, thereby driving the synchronous expression of OSK and OGL, achieving a transient, coordinated, and controllable partial reprogramming of the five OSKGL factors. After the intervention window ends, Dox is withdrawn, and rtTAX loses its ligand and reverts to an inactive state, with OSKGL expression shut down. Compared to the tet-off (tTA) system, the tet-on system represented by rtTAX is strictly closed under Dox-free conditions and can only be activated after Dox administration, fundamentally eliminating the risk of relying on continuous Dox administration to maintain gene silencing, making it more suitable for long-term preclinical research and potential clinical translational applications.

[0040] 1.1.1 Design and Functional Advantages of rtTAX

[0041] rtTAX (reverse tetracycline transactivator X) is a novel modified variant obtained by introducing three site-directed mutations into rtTA2s-M2 (hereinafter referred to as rtTA2, Urlinger et al., Proc Natl Acad Sci USA, 2000, known prior art). rtTA2 is a publicly disclosed classic reverse tetracycline transactivator. Its TetR DNA-binding domain contains known functional mutations such as H12L, E19G, A56P, and D148E, possessing certain doxycycline-responsive activity. However, it has two major limitations: high in vivo basal leakage expression (it can still activate about 15%-25% of TRE-driven expression even without Dox) and low in vivo Dox-sensing sensitivity (EC50 about 8-12 ng / mL), which restricts its application in in vivo gene therapy requiring precise regulation.

[0042] This invention introduces the following three independent point mutations into rtTA2 by combining computer-aided protein structure simulation (based on the TetR-tetracycline cocrystal structure, PDB ID: 1BJY) and directed evolutionary screening: Mutation 1 (T45A, threonine → alanine, located in the α3 region of the N-terminal α-helix of TetR): The hydroxyl side chain at the T45 site forms hydrogen bonds with neighboring amino acids in the ligand-free (apo) state, stabilizing a conformation that is conducive to non-specific binding of DNA; after introducing the more hydrophobic alanine, the conformational stability in the apo state is reduced, thereby reducing the non-specific binding of rtTAX to the TRE promoter under Dox-free conditions and effectively reducing background leakage expression.

[0043] Mutant 2 (S95N, serine → asparagine, located in the loop region at the edge of the TetR ligand binding pocket): The polar side chain at the S95 site can form additional hydrogen bonds with the C1 carbonyl group of the Dox molecule, thereby increasing the binding affinity of rtTAX to Dox; after introducing asparagine with a longer side chain, the hydrogen bond force is enhanced, allowing rtTAX to be fully activated at lower Dox concentrations, resulting in an EC50 reduction from approximately 10 ng / mL for rtTA2 to approximately 3–5 ng / mL.

[0044] Mutation 3 (L138V, leucine → valine, located in the hydrophobic core β-sheet region of TetR): The L138 site is located near the TetR dimer interface. The larger hydrophobic side chain of leucine maintains a protein dimer conformation favorable for TRE binding in the Dox-free state. After being replaced by the smaller valine side chain, the hydrophobic accumulation at the dimer interface in the apo state is weakened, reducing the ability of rtTAX in the ligand-free state to recruit to the TRE promoter region, further synergistically reducing leakage expression. At the same time, the L138V mutation does not interfere with the Dox-binding pocket conformation and does not affect the ligand-induced activation efficiency.

[0045] The synergistic effect of the above three mutation combinations enables rtTAX to achieve the following compared to rtTA2: (i) a reduction of approximately 85% in in vivo basal leakage expression (TRE-driven luciferase reporter gene activity ≤ 15% of rtTA2 leakage level under Dox-free conditions); (ii) a reduction of the Dox half-maximal activation concentration (EC50) to 3–5 ng / mL (rtTA2 EC50 is approximately 10 ng / mL, a reduction of approximately 2–3 times); and (iii) a maximum induction fold ≥ 120% of rtTA2 in the fully activated state, ensuring that the expression intensity of the five factors is not lower than that of the control system during the intervention window. The above three mutation sites (T45, S95, L138) do not overlap with the mutation sites used in other rtTA modified variants reported in existing literature in terms of amino acid sequence and structural position, constituting an independent technical contribution of this invention.

[0046] The full-length rtTAX amino acid sequence is approximately 242–248 amino acids (preferably approximately 248 amino acids, based on the rtTA2 sequence, with a triple mutation of T45A / S95N / L138V). Its encoding DNA sequence is approximately 747–780 bp in length (preferably approximately 747 bp). After codon optimization (optimized for human cell expression, CAI ≥ 0.85), it was synthesized by a gene synthesis company, with Kozak sequences (GCCACCATG) and stop codons added at both ends, and cloned downstream of the pAAV backbone UBC promoter.

[0047] 1.2 Technical Specifications for Carrier Construction

[0048] Construction of the pAAV8-TRE3G::OSK vector: Using human Oct4 (POU5F1, NM_002701), Sox2 (NM_003106), and Klf4 (NM_004235) cDNA as templates, the coding sequences of each factor were amplified by PCR and sequentially cloned into a modified pAAV backbone containing a TRE3G promoter (the backbone contained a short expression cassette to ensure that the total inserted fragment did not exceed 4.7 kb); a T2A sequence (EGRGSLLTCGDVEENPGP, 18 amino acids, encoding 54 bp, as shown in SEQ ID NO: 1) was inserted between Oct4 and Sox2, and a P2A sequence (ATNFSLLKQAGDVEENPGP, 19 amino acids, encoding 57 bp, as shown in SEQ ID NO: 2) was inserted between Sox2 and Klf4; the above self-cleaving peptide sequences all used compact preferred variants, which can also be replaced with functionally equivalent extended variants containing an N-terminal GSG flexible linker; WPRE3 (300) was sequentially linked to the 3' end. (221 bp optimized version) and SV40 polyA signal (221 bp); AAVITR sequence (145 bp×2) is retained at both ends.

[0049] Construction of the pAAV8-TRE3G::OGL vector: Using human Glis1 (NM_147193) and Lin28A (NM_024674) cDNA as templates, the vector was constructed using the same method. An E2A sequence (CTNYALLKLAGDVESNPGP, 19 amino acids, encoding 57 bp, as shown in SEQ ID NO: 3) was inserted between Glis1 and Lin28A. Alternatively, it could be replaced with a functionally equivalent extended variant containing an N-terminal extension (such as QCTNYYKTRAGGSGEGRGSLLTCGDVEENPGP, 32 amino acids, encoding 96 bp, as shown in SEQ ID NO: 4).

[0050] pAAV8-UBC::rtTAX-WPRE3-SV40pA vector: The rtTAX coding sequence (T45A / S95N / L138V triple mutation, codon optimized version) synthesized in this invention is cloned into the pAAV skeleton driven by the UBC promoter, and WPRE3 and SV40polyA are configured.

[0051] All plasmids were validated for full length using Sanger sequencing, and high-purity plasmids were prepared using the Qiagen EndoFree Maxi Kit for virus production.

[0052] 1.3 AAV8 Virus Production and Quality Control

[0053] Three AAV8 vectors were produced using a three-plasmid co-transfection HEK293T cell system. HEK293T cells (ATCCCRL-3216) were cultured in 15 cm culture dishes (10% FBS / DMEM, 37℃ / 5% CO2) at a seeding density of 1.5 × 10⁻⁶ cells / year. 7 Cells were cultured in dishes until confluence reached approximately 60%, then co-transfected with three plasmids (plasmid mass ratio: transfer plasmid:pHelper:pAAV-RC8=1:1:1, total plasmid amount 45μg / dish, polyethyleneimine (PEI) mediated, PEI:DNA=3:1, w / w). 72 hours post-transfection, cells and culture supernatant were collected and lysed by freeze-thaw cycles (-80℃ / 37℃ alternating 3 times). The cells were then purified by ultracentrifugation (350,000×g, 2.5 h, 4℃) using a discontinuous density gradient of iodixanol (OptiPrep, Sigma) at a density of 16% / 25% / 40% / 60%. 40% / 60% interfacial fractions were collected and concentrated by ultrafiltration with PBS / 5% sorbitol (Amicon Ultra-15, 100 kDa MWCO).

[0054] Viral titers were precisely quantified using digital droplet PCR (ddPCR, targeting the WPRE sequence). Working titer standards were: pAAV8-TRE3G::OSK 2.0–3.0 × 10¹³ vg / mL, pAAV8-TRE3G::OGL 2.0–3.0 × 10¹³ vg / mL, and pAAV8-UBC::rtTAX 2.5–3.0 × 10¹³ vg / mL. Viral purity was verified using SDS-PAGE silver staining (VP1:VP2:VP3 ratio approximately 1:1:10), and endotoxin levels, as detected by LAL, should be below 1 EU / mL. Packaging integrity verification: Considering the OSK vector's position near the upper limit of AAV8 packaging capacity, dual quantification was performed after virus production using digital droplet PCR (ddPCR) targeting both the 5' and 3' ITR sequences. The 5' / 3' titer ratio was confirmed to be within the range of 0.8–1.2 (indicating the absence of significantly truncated products). Simultaneously, the genomic integrity of the transfer plasmid was verified by full-length expression cassette PCR amplification (designing a long primer pair spanning the 5' end of Oct4 to the 3' end of KLF4, with an expected amplification product of approximately 3.6 kb). If the 5' / 3' titer ratio was <0.8 or truncated bands appeared in the full-length PCR, the regulatory elements needed to be further compressed (e.g., replacing the full-length WPRE3 of approximately 250 bp with a mini-WPRE of approximately 170 bp, or replacing the standard SV40 polyA of approximately 200 bp with a compact SV40 polyA of approximately 125 bp) before repackaging and verification.

[0055] Functional validation (rtTAX specificity validation): HEK293T cells were co-infected with three viruses (MOI 1×10⁻⁶ each). 4 The Dox group was divided into four groups: (i) Dox completely removed (0 ng / mL); (ii) low Dox group (5 ng / mL); (iii) medium Dox group (50 ng / mL); and (iv) high Dox group (1000 ng / mL).

[0056] After 48 hours of culture, Western blot analysis was performed on the expression of Oct4, Sox2, Klf4, Glis1, and Lin28 proteins in each group. Dose-response curves were established to confirm that the EC50 fell within the range of 3-5 ng / mL, and that the expression levels of all five proteins in the 0 ng / mL group were below the detection limit (verifying the low-leakage characteristics of rtTAX). A control group (with the same MOI and treatment) was also set up to compare and verify the functional improvement of rtTAX in terms of low leakage and high sensitivity. Virus solution was aliquoted (single-use volume, 10 μL / tube) and stored at -80℃ to avoid repeated freeze-thaw cycles.

[0057] 1.4 Preparation and Preoperative Management of Laboratory Animals

[0058] Nine-month-old B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo / Mmjax (APP / PS1) transgenic AD model mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. or an authorized agent of Jackson Laboratory in China, half male and half female, weighing 25–35 g) and age-matched wild-type C57BL / 6J control mice were used. Dox was not administered before surgery or during routine feeding conditions, rtTAX remained in an inactive state, and TRE3G-driven OSKGL expression was naturally kept off or at extremely low leakage levels (below the detection limit) under basal conditions. Animals were housed in a standard SPF barrier facility (12-hour light-dark cycle, free access to food and water). For 2–3 days prior to surgery, the same laboratory personnel acclimated the animals to daily touch to reduce stress during surgery and behavioral testing. All animal experimental procedures were approved by the institution's laboratory animal ethics committee (ethics approval number: IACUC-343).

[0059] Before use, thaw the three viral solutions (pAAV8-TRE3G::OSK, pAAV8-TRE3G::OGL, and pAAV8-UBC::rtTAX) slowly on ice and mix them in equal volumes (volume ratio 1:1:1). The final working solution should contain approximately 0.8–1.0 × 10¹³ vg / mL of each virus. Gently mix before injection, keep on ice, and use within 2 hours.

[0060] 1.5 Stereotactic intracranial injection

[0061] Animals were induced to undergo general anesthesia by intraperitoneal injection of an anesthetic mixture (fentanyl 0.05 mg / kg + midazolam 5 mg / kg + metopridine 0.5 mg / kg), and surgery was performed after the corneal reflex disappeared. The hair on the top of the head was shaved, and an analgesic mixture (lidocaine 6 mg / kg + bupivacaine 2.5 mg / kg) was injected subcutaneously. The animal was fixed in a small animal stereotaxic apparatus (Kopf Instruments or equivalent precision device). After disinfecting the surgical area with 75% alcohol and iodine, the scalp was incised approximately 1.5 cm along the midline of the sagittal suture. The skull was exposed by rinsing with saline. The anterior fontanelle (Bregma) and lambda suture were used as dual reference points to correct the skull level to an error of <0.1 mm. A skull hole (approximately 0.5 mm in diameter) was drilled on the skull surface of the target brain region using a dental drill. A drawn borosilicate glass micropipette (approximately 15-20 μm outer diameter) was connected to a microinfusion pump (Harvard Apparatus), and the triviral mixture was injected at a constant rate of 0.2 μL / min. Dentate gyrus (DG) injection coordinates (bilateral): AP -1.9 mm, ML ±1.3 mm, DV -2.0 mm (measured from the skull surface); injection volume per side 300 nL, containing approximately 3 × 10⁻⁶ OSK virus. 9 Approximately 3 × 10⁻⁶ VG and OGL viruses 9 Approximately 3 × 10vg and rtTAX viruses 9 vg.

[0062] Medial prefrontal cortex (mPFC) injection coordinates (bilateral): AP +2.0 mm, ML ±0.35 mm, DV -2.25 mm; injection volume per side 250 nL, content converted proportionally.

[0063] After injection, the micropipette was left in place at the injection site for 5 minutes to prevent backflow. The needle was then slowly withdrawn to the skull surface at a rate of 0.3 mm per minute. The bone hole was sealed with bone wax, and the scalp was sutured continuously with 4-0 absorbable sutures. Atipametrexed (2.5 mg / kg) was administered intraperitoneally for reversal anesthesia. The animal was then placed on a heating pad (37°C) for recovery, and acetaminophen (2 mg / mL, for 3 consecutive days) was added to its drinking water for analgesia. Animals with a transduction efficiency of less than 2% or significant deviation from the injection coordinates (confirmed by postoperative histology) were considered invalid individuals and excluded from further analysis.

[0064] 1.6 OSKGL Expression Induction and Regulation of the Safe Therapeutic Window

[0065] Animals recovered for at least 2 weeks after AAV injection, during which time Dox was not administered, and rtTAX remained in an inactive state to ensure that OSKGL did not develop basal expression during the transduction and colonization period. During the pre-defined intervention window, Dox (0.2 mg / mL, dissolved in 5% sucrose-containing drinking water) was administered via oral fluid to initiate the OSKGL induction expression window. The intervention window was set at 2 days. During this period, taking advantage of rtTAX's high Dox sensitivity (EC50 approximately 3–5 ng / mL), a precise dose of Dox (1 mg / kg / day, twice daily) was administered via gavage for fine-tuning, achieving gradual OSKGL induction at concentrations below the complete activation level, thus avoiding over-reprogramming caused by acute overexpression of Factor V. After the intervention window ended, Dox was removed from the drinking water (replaced with 5% sucrose-containing drinking water without Dox), and rtTAX lost its ligand and returned to an inactive state, shutting down OSKGL expression.

[0066] During the intervention window (days 1 and 2) and on days 3, 7, and 14 after the window ends, blood was collected via tail vein (0.1 mL each time), and peripheral blood mononuclear cells were isolated for DNA methylation clock detection to dynamically calculate the epigenetic age reversal margin (RM value). The intervention protocol was adjusted dynamically based on the RM value: if RM < 60%, the next intervention window period could be appropriately extended (maximum increase of 1 day) or the dose of Dox administered orally could be slightly increased after a safety assessment; if RM > 75%, full-dose Dox was immediately resumed via drinking water and the intervention was suspended until RM returned to the 60%-75% safety window.

[0067] 1.7 Transduction efficiency verification and safety assessment

[0068] Animals were sacrificed at the end of the experiment, and coronal frozen sections (30 μm) of the target brain region were taken for the following tests: Transduction efficiency verification: AAV-OSK transduction was verified by dual-color immunofluorescence staining with anti-Sox2 antibody (Cell Signaling Technology, #23064, 1:500, Alexa Fluor 488 labeled secondary antibody) and anti-Klf4 antibody (R&D Systems, AF3158, 1:400, Alexa Fluor 568 labeled secondary antibody), AAV-OGL transduction was verified by anti-Lin28A antibody (Cell Signaling Technology, #8641, 1:500, Alexa Fluor 647 labeled secondary antibody), and the integrity of all five factors transduction was verified by anti-Glis1 antibody. The expected effective transduction group had a Klf4 / Sox2 double-positive cell ratio ≥25% in the DG and mPFC target areas and a Lin28A positive cell ratio ≥20% (with the total number of DAPI-positive cells as the denominator).

[0069] Cellular identity preservation verification: The preservation of neuronal cellular identity after OSKGL intervention was verified using anti-Prox1 antibody (DG granule cell identity marker, Abcam, ab101851, 1:1000) and anti-Ctip2 antibody (mPFC deep neuronal marker, Abcam, ab18465, 1:400). It was expected that the expression levels of each marker would not be lower than those of the non-transduced control neurons in the same region, demonstrating that the OSKGL combination without c-Myc can achieve epigenetic remodeling without inducing neuronal dedifferentiation.

[0070] In vivo validation of low leakage of rtTAX: A positive control group was set up with only AAV-rtTAX injection (only pAAV8-UBC::rtTAX was injected, without OSK+OGL virus, and Dox was maintained in drinking water throughout the experiment). At the end of the experiment, brain tissue was collected for Western blot detection of Oct4, Sox2, and Klf4 proteins. The expected expression levels of the three proteins were all below the detection limit (<0.5 ng / mg total protein), demonstrating the strict controllability of the rtTAX and TRE3G combined system and the effective inhibition of background leakage by the three rtTAX mutations.

[0071] Safety testing: Ki67 immunofluorescence staining (Abcam, ab16667, 1:200) was used to systematically scan all regions of the whole brain, and the Ki67-positive cell detection rate in the expected therapeutic window dose group was 0%; H&E staining (whole brain slides) was used to assess the damage and inflammatory infiltration of the injection site and surrounding brain regions; complete blood cell count (experimental endpoint) and serum pro-inflammatory cytokine levels (IL-6, TNF-α, IFN-γ, ELISA) were used to assess systemic safety; anti-AAV8 neutralizing antibody titer (comparison of baseline before injection and experimental endpoint) was used to assess the immunogenicity of the vector.

[0072] 1.8 Transnasal delivery alternatives

[0073] For subjects unsuitable for intracranial stereotactic injection, this invention further provides a nasal targeted delivery protocol as a supplementary approach: The above-mentioned three AAV8 virus mixtures (titer and ratio as in Section 1.4) are combined with a mucosal penetration enhancer (0.05% sodium hyaluronate, w / v) to prepare a nasal drip formulation. Under mild anesthesia (2% isoflurane, inhalation, 2 minutes), the virus is slowly dripped through the nostrils (5 μL per nostril, total 10 μL, total viral load approximately 1 × 10¹¹ vg / animal), once a week for four consecutive weeks, to achieve non-invasive targeted delivery of the virus to the olfactory bulb, hippocampus, and prefrontal cortex via the olfactory nerve bundle and trigeminal nerve pathway. The OSKGL expression induction protocol, safety therapeutic window monitoring protocol, and endpoint assessment protocol for the nasal administration group are the same as those for the intracranial injection group. The expected effective transduced cell ratio in the target area is 20%–40% of that in the intracranial injection group, suitable for low-intensity reprogramming intervention needs in the early stages of AD and MCI.

[0074] 1.9 Statistical Analysis

[0075] All data were statistically analyzed using GraphPad Prism 10. Behavioral data (path length, swimming speed) were analyzed using two-way repeated measures ANOVA; quadrant preference and rigid response were analyzed using one-way ANOVA; changes in swimming strategy proportions were analyzed using the logistic regression odds ratio (OR) test; cell count and fluorescence intensity data were analyzed using the unpaired two-tailed Student's t test or the Mann-Whitney U test; comparisons among multiple groups were performed using one-way ANOVA with Tukey multiple comparison correction. Significance threshold: p < 0.05 ( p<0.01 p<0.001 p<0.0001 All data are expressed as mean ± standard error (Mean ± SEM).

[0076] A quantitative detection system based on the DNA methylation clock of peripheral samples; this detection system is the core of the present invention for achieving non-invasive and accurate monitoring.

[0077] (1) Sample collection: Cerebrospinal fluid or peripheral venous blood were collected from the subjects before intervention (baseline) and periodically after intervention.

[0078] (2) DNA extraction and methylation analysis: Circulating cell-free DNA or mononuclear cell genomic DNA is extracted and analyzed using high-precision methylation chips or targeted sequencing.

[0079] (3) Epigenetic age calculation: Input the methylation data into a blood or brain tissue-specific epigenetic clock model validated by an AD patient cohort, and output "predicted epigenetic age".

[0080] (4) Calculation of “Age Acceleration Value” and “Reversal Amount”: Epigenetic age acceleration = predicted epigenetic age - actual chronological age.

[0081] Disease-related acceleration difference = Average age acceleration of AD patients - Average age acceleration of healthy peers.

[0082] Reversal magnitude = [(baseline EAA - post-intervention EAA) / disease-related acceleration difference] × 100%.

[0083] Experimental results: I. Results of Cognitive Function Behavioral Tests

[0084] 1.1 Morris Water Maze Test

[0085] On day 28 after OSKGL nucleic acid composition intervention, mice in each group underwent five consecutive days of orientation and navigation training and a space exploration experiment on day 6. Experimental groups: wild-type control group (WT-Vehicle, n=12), APP / PS1 model control group (AD-Vehicle, n=11, excluding one individual with floating behavior), APP / PS1+OSKGL treatment group (AD-OSKGL, n=12), and APP / PS1+OSKGL high-dose group (AD-OSKGL-High, n=9, excluding one individual with continuous postoperative weight loss, RM>75% subgroup).

[0086] Table 1. Morris water maze positioning and navigation training escape latency (seconds, Mean±SEM)

[0087] Note: Two-way repeated measures ANOVA, group × time interaction effect F(12,156) = 3.94, p = 0.00004, Greenhouse-Geisser correction ε = 0.72. Post hoc Tukey correction.

[0088] Table 2 Morris Water Maze Space Exploration Experiment (Day 6, Platform Removal, 60 seconds)

[0089] Note: There was no significant difference in swimming speed among groups (F(3,40) = 0.15, p = 0.93), excluding the interference of motor function differences interference.

[0090] 1.2 New Object Recognition Test

[0091] The tests were conducted on day 30 after the intervention, at two time points: 1 hour and 24 hours later. Individuals with a total exploration time of less than 15 seconds were excluded (1 AD-Vehicle and 1 AD-OSKGL-High were excluded).

[0092] Table 3 New Object Recognition Index (DI = (T_novel - T_familiar) / (T_novel + T_familiar))

[0093] Note: p < 0.001 vs WT; p=0.0048, p = 0.014 vs AD-Vehicle. There was no difference in total exploration time among groups (p = 0.68).

[0094] 1.3 Spontaneous Alternation Experiment in Y-shaped Maze

[0095] Table 4. Spontaneous Alternation Rate and Total Number of Arm Entry in the Y-shaped Maze

[0096] Note: Chance level is 50%. There was no significant difference in the total number of arm entries among groups (p = 0.81).

[0097] II. Dynamic monitoring data of epigenetic age acceleration value (EAA) and reversal magnitude (RM)

[0098] Based on the peripheral blood mononuclear cell DNA methylation clock (Horvath multi-tissue clock calibrated in an AD mouse cohort, 353 CpG sites targeted bisulfite sequencing). Background parameters: mean EAA of 9-month-old WT mice = +0.3±0.8 weeks (n=12); mean EAA of 9-month-old APP / PS1 baseline = +8.7±1.2 weeks (n=22); disease-related acceleration difference = 8.4 weeks.

[0099] Table 5. Dynamic changes in EAA and RM in the AD-OSKGL treatment group (n=12)

[0100] Note: Paired t-test. RM reached a plateau at the end + 7d (~70%), maintained 73.8% at + 28d, and decreased to 58.3% at + 56d.

[0101] Table 6. Dynamic changes of EAA and RM in the AD-OSKGL-High high-dose group

[0102] Note: The peak value of RM was 97.7%, significantly exceeding the upper limit of the 75% safety window.

[0103] 2.3 Dose-response relationship between RM value and efficacy indicators

[0104] All OSKGL intervention animals (n=22) were divided into four subgroups based on their RM value at end +14 days.

[0105] Table 7. Different RM intervals and multidimensional efficacy indicators

[0106] Note: vs AD-Vehicle, one-way ANOVA + Dunnett correction. Aβ42 and p-Tau in the >75% group still showed significant decrease ( p=0.015, p = 0.0072), but the Iba1+ area and IL-6 showed a rebound increase, and there was no significant improvement in cognitive indicators. improvement.

[0107] 2.4 Correlation analysis between RM values ​​and cognitive / pathological indicators

[0108] Individuals with RM in the range of 25-80% (n=17) were included to assess linear segment associations.

[0109] Table 8 Pearson Correlation Analysis

[0110] Note: After including all 22 animals, the quadratic coefficient of MWM latency vs RM was significant (β 2=+0.18, p= 0.008), supporting the non-linear threshold effect.

[0111] III. Quantitative data on immunofluorescence staining transduction efficiency

[0112] The experimental endpoint was coronal frozen sections (30 μm), with 3 sections per brain region × 5 randomized 400 × field of view (15 field of view / animal / brain region), and DAPI as the denominator. Double-blind counting was conducted by two participants, with ICC = 0.91.

[0113] Table 9. Transduction efficiency of the hippocampal dentate gyrus (DG) (AD-OSKGL group, n=12)

[0114] Table 10 Transduction efficiency of medial prefrontal cortex (mPFC) (AD-OSKGL group, n=12)

[0115] Note: The double-positive rate of Sox2+ / Klf4+ in the DG region was 30.8%, exceeding the preset threshold ≥ 25%, and the positive rate of Lin28A+ was 23.8%, exceeding the threshold ≥ 20%. The data in the mPFC region was similar. Excluded individuals: #07 (DG double-positive rate 8.2%) and #11 (6.5%), and postoperative histology confirmed that the injection coordinates deviated. confirmation of injection coordinate deviation.

[0116] Table 11 Transduction efficiency of the nasal delivery group (AD-OSKGL-IN group, n=8)

[0117] Note: The target area transduction rate of the intranasal group was about 25 - 29% of that of the intracranial injection group, within the expected range (20 - 40%).

[0118] IV. Cell Identity Preservation and Verification Data

[0119] Sox2+ / Klf4+ positive neurons in the transduction area were paired with untransduced neighboring neurons (distance <100μm) in the same slice for comparison, 120 pairs / group.

[0120] Table 12. Preservation of Prox1 expression in the hippocampal DG region (AD-OSKGL group, n=12)

[0121] Table 13. Ctip2 expression retention in the mPFC region (AD-OSKGL group, n=12)

[0122] Note: All comparisons did not reach statistical significance (p > 0.05), confirming that the neuronal cell identity was intact after OSKGL intervention preserved.

[0123] Table 14 Screening of dedifferentiation markers (AD-OSKGL group, n=12)

[0124] V. rtTAX Low Leakage Verification Data

[0125] 5.1 Western Blot Detection Methods and Limit of Quantitation Calibration

[0126] A standard curve was established using a chemiluminescence imaging system (Bio-Rad ChemiDoc MP) with recombinant protein standards. The limits of detection (LOD, S / N=3) and quantitation (LLOQ, S / N=10, CV<20%) were determined as follows.

[0127] Table 15 LOD and LLOQ values ​​for each protein

[0128] Table 16. Quantitative results of in vivo Western Blot (hippocampal tissue)

[0129] Note: ND = not detected. All proteins in the rtTAX-only group were < LOD. Some individuals in the triple-vector / no Dox group could detect signals but were below the LLOQ and could not be accurately quantified; taking the LLOQ as the worst-case scenario, the upper limit of the leakage rate of each protein was < 2%. Note: rtTAX EC50 = 3.8 ± 0.6 ng / mL (four-parameter logistic fit), rtTA2 EC50 = 9.7 ± 1.2

[0130] Table 17 rtTAX vs rtTA2 in vitro luciferase reporter gene dose-response (HEK293T, n=3 independent experiments)

[0131] ng / mL. The leakage of rtTAX at 0 ng / mL was 15.9% of rtTA2. The maximum activity of rtTAX in the saturated state was 124% of rtTA2. Note: The Ki67+ in the target area of the AD-OSKGL group was 0, showing no difference from WT (Fisher's exact test p = 1.0). Eight Ki67+ were detected in the target area of the AD-OSKGL-

[0132] VI. Safety Test Data

[0133] 6.1 Whole brain scan with Ki67 proliferation marker

[0134] Whole brain coronal sections (300 μm interval, approximately 40 sections / brain), Ki67 immunofluorescence (Abcam ab16667, 1:200), double-blind scanning, κ=0.95.

[0135] Table 18 Detection of Ki67 positive cells

[0136] High group, including 5 in the DG hilus region (abnormal SGZ position) and 3 in the deep layer of the mPFC, showing an ectopic distribution. Note: There were 8 Ki67+ detected in the target area of the AD-OSKGL-High group, including 5 in the DG hilus region (abnormal SGZ position) and 3 in the deep layer of the mPFC, showing an ectopic distribution.

[0137] 6.2 Whole-brain H&E histopathological assessment

[0138] Independent pathologists used a double-blind scoring system on a 0-4 scale (0=normal, 1=very mild / focal, 2=mild / multifocal, 3=moderate / diffuse, 4=severe), with a weighted κ=0.88.

[0139] Table 19 H&E Semi-Quantitative Scoring

[0140] Note: The AD-OSKGL group was significantly superior to the AD-Vehicle group in terms of neuronal degeneration and glial proliferation. High-dose group target... The inflammation score in the area was high (1.9 vs 0.8, p=0.018).

[0141] 6.3 Complete blood count (CBC)

[0142] Table 20 Experimental endpoint: Complete blood cell count

[0143] Note: All parameters were within the normal range, and there were no statistically significant differences between the groups.

[0144] 6.4 Serum pro-inflammatory cytokines (ELISA)

[0145] Table 21 Serum cytokine levels (pg / mL)

[0146] Note: ###p<0.001, ##p<0.01 vs WT. All four pro-inflammatory factors were significantly decreased in the AD-OSKGL group; IL-10 The recovery has not been significant.

[0147] 6.5 Anti-AAV8 neutralizing antibody titer

[0148] Table 22 Neutralizing antibody titers

[0149] Note: High titers of neutralizing antibodies were produced in all intracranial injection groups, while the nasal injection group produced approximately 1 / 4 of the titers in the intracranial group.

[0150] VII. Supplementary Inspection Items

[0151] 7.1 Quantitative analysis of Aβ and p-Tau pathological proteins

[0152] Table 23 Aβ42 and p-Tau in the hippocampus (ELISA + immunohistochemistry)

[0153] Note: In the AD-OSKGL group, soluble Aβ42 decreased by 67% and AT8 decreased by 68%. There was no difference in total Tau between groups; intervention selectivity was observed. Reduced phosphorylation modification. The high-dose group showed a reduction of 35-52%, significantly weaker than the therapeutic window group (Aβ42 group p=0.028, AT8 group p=...). 0.015）。

[0154] 7.2 Intracerebral neuroinflammatory markers

[0155] Table 24 Quantitative analysis of hippocampal glial cell activation

[0156] 7.3 Quantitative analysis of synaptic markers

[0157] Table 25 Hippocampal CA1 synaptic proteins (Western Blot, β-actin normalized, WT=1.00)

[0158] 7.4 Autophagy-lysosomal pathway

[0159] Table 26 Autophagy-related proteins (Western Blot, β-actin normalized)

[0160] Note: p<0.05, p<0.01, p<0.001, p<0.0001 vs AD-Vehicle. High dose The LC3-II / I ratio increased to 0.68 (above the WT level of 0.45), while p62 decreased to 0.78 (below the WT level of 1.00).

[0161] 7.5 Genomic stability assessment

[0162] Table 27 DNA damage markers (immunofluorescence, DG region, n=6 / group, 50 cells / animal)

[0163] Note: p<0.0001, p=0.0005 vs AD-Vehicle. †p<0.05 vs AD-Vehicle (square (The levels were elevated). In the high-dose group, γH2AX (5.2) and 53BP1 (4.1) were both higher than in the AD-Vehicle group (3.5 and 2.8).

[0164] VIII. Summary of Core Conclusions

[0165] Table 28 Summary of Experimental Results for Each Dimension

[0166] In summary, when the epigenetic age reversal rate is strictly controlled within 60%-75%, OSKGL nucleic acid composition intervention can significantly improve the cognitive function of APP / PS1 transgenic mice, effectively clear Aβ and p-Tau pathological proteins, inhibit neuroinflammation, and restore synaptic protein expression, while no abnormalities were observed in proliferation markers, genomic stability, and systemic safety indicators. When the reversal rate exceeds 75%, the cognitive benefit diminishes, and safety signals such as ectopic Ki67+ cells in the target area, increased γH2AX foci, and rebound neuroinflammation appear.

Claims

1. The application of a nucleic acid composition based on quantitative partial reprogramming in the preparation of drugs for the prevention and treatment of Alzheimer's disease, characterized in that, The composition comprises nucleic acid constructs encoding Oct4, Sox2, Klf4, Glis1, and Lin28 proteins.

2. The application according to claim 1, characterized in that, The nucleic acid construct does not encode the c-Myc protein.

3. The application according to claim 1, characterized in that, The nucleic acid construct is carried in a neural tropism delivery vector.

4. The application according to claim 1, characterized in that, The neural directional delivery vector is a recombinant adeno-associated virus vector, a nasal targeted delivery system, or a combination thereof.

5. The application according to claim 1, characterized in that, The nucleic acid construct contains inducible gene expression regulatory elements.

6. The application according to claim 5, characterized in that, The inducible gene expression regulatory element is a tetracycline-inducible expression system.

7. The application according to claim 6, characterized in that, The nucleic acid construct comprises a combination of the following three recombinant adeno-associated virus vectors: (1) A first recombinant adeno-associated virus vector containing nucleic acid sequences encoding Oct4, Sox2 and Klf4 proteins that are controlled by an inducible promoter; (2) A second recombinant adeno-associated virus vector, comprising the nucleic acid sequences encoding Glis1 and Lin28 proteins controlled by the inducible promoter; and (3) The third recombinant adeno-associated virus vector contains a nucleic acid sequence encoding a reverse tetracycline transactivator linked by a constitutive promoter.

8. The application according to claim 7, characterized in that, In the first recombinant adeno-associated virus vector, the nucleic acid sequences encoding Oct4, Sox2, and Klf4 proteins are tandemly linked by self-cleaving peptide sequences; in the second recombinant adeno-associated virus vector, the nucleic acid sequences encoding Glis1 and Lin28 proteins are tandemly linked by self-cleaving peptide sequences.

9. The application according to claim 5, characterized in that, The inducible promoter is the TRE3G promoter, and the constitutive promoter is the human ubiquitin C promoter; the reverse tetracycline trans-activator is a modified rtTAX, which is a variant obtained by introducing three point mutations, T45A, S95N and L138V, on the rtTA2s-M2 amino acid sequence.

10. A pharmaceutical composition for the treatment of Alzheimer's disease, characterized in that, It includes the nucleic acid construct according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier or excipient.

Images