Table 2.

Applications of gene therapy vectors

Vector type	Name	Advantages	Disadvantages	Applications of gene therapy in ALS
Viral vectors	Ad	Safe and effective gene vectors in early gene therapy studies	1. Ad can easily trigger strong immune and inflammatory responses in patients 2. The duration of transgene expression is very limited	Ad-GDNF therapy can prevent the loss of motor neurons in a transgenic mouse model with SOD1 mutations by preserving the survival p-Akt (phosphorylated Akt) signaling without affecting caspase activation [26]
	AAVs	1. Do not elicit significant immune responses or cause disease 2. Effectively infect mammalian cells without the need for helper viruses 3. Extensively studied with broad application prospects	1. Traditional AAVs have low penetration ability through the blood–brain barrier 2. Traditional AAVs have low central nervous system- targeting specificity Some AAVs have high peripheral tissue targeting affinity, leading to off-target effects 3. Novel AAVs with high central nervous system affinity or central administration methods can cause internal CNS diffusion, leading to various central adverse effects 4. Previous exposure to related viruses can result in memory humoral immune elimination of AAV upon subsequent use	1. Subpial delivery mediated by AAV is a currently safer gene therapy technique under development [29, 30] 2. AAV9-SynCav1 delivered via spinal subpial delivery preserves neuro-muscular function and α-motor neurons in the ventral horn of double transgenic (SynCav1 TG/hSOD1G93A) mice [29] 3. Subpial delivery of AAV9-shRNA-SOD1 in SOD1G37R mouse models shows extensive gene silencing, significant preservation of motor function and α-motor neurons, and halts disease progression in symptomatic mice [30] 4. AAV-IGF-1 promotes IGF-1 synthesis in spinal motor neurons of ALS mice carrying the hSOD1 mutation, successfully prolonging mouse survival [31] 5. AAV-NDNF, administered early after symptom onset, improves motor performance and weight maintenance; mid-term administration enhances motor abilities, and late-stage injections extend lifespan [32] 6. AAV-mediated CRISPR-Cas9 gene editing demonstrates gene regulation capabilities in mouse cochleae, expanding the potential applications of AAVs in gene therapy, with future opportunities for ALS treatment [34]
	Lentiviral Vectors	Can achieve long-lasting transgene expression at low titers	1. Difficulty in crossing the blood–brain barrier, with gene transduction primarily limited to areas near the injection site 2. Retroviral vectors may integrate DNA into the host chromosome, potentially leading to abnormal expression and tumor formation	1. The use of lentiviral vectors expressing VEGF via retrograde transport at nerve terminals has been shown to protect neurons, significantly delaying the progression of ALS in SOD1G93A mutant mice [38] 2. Lentiviral vectors carrying shRNA, delivered to primary neuronal cells, silence SOD1 expression, ultimately delaying the onset of symptoms and extending survival in SOD1-ALS mice [17]
Non-viral vectors	Liposomes	1. Biocompatible, allowing delivery of lipophilic or hydrophilic therapeutic agents, effectively penetrating the blood–brain barrier for targeting the central nervous system 2. Novel lipid nanoparticles are smaller than traditional ones, exhibit higher in vivo stability, greater therapeutic payload capacity, lower immunogenicity, enhanced nucleic acid encapsulation, and improved in vivo release capabilities	1. Short duration in vivo, stability needs improvement 2. High precision required for liposome formulations; incompatibility with drugs can lead to drug leakage or ineffectiveness	1. Liposomes as carriers for transporting riluzole and verapamil effectively penetrate the central nervous system for ALS treatment [43] 2. CaP lipid nanoparticles efficiently and safely deliver ASOs, reducing levels of mutant SOD1 in motor neurons of ALS mice [45]
	Naked DNA/RNA Vectors	1. Simple and straightforward use, with strong controllability 2. Relatively simple and convenient preparation and purification processes 3. Low immunogenicity, avoiding carrier-related side effects	1. Low transfection efficiency 2. Insufficient selectivity for specific cells 3. Poor stability in vivo	The gene encoding TTC was cloned into the pcDNA eukaryotic expression vector and intramuscularly injected into transgenic SOD1G93A mouse models, significantly delaying symptom onset and functional deficits while improving the survival rate of spinal motor neurons [46]
	Protein Vectors	1. High specificity of targeting, strong cellular penetration capability 2. Excellent drug release regulation	1. Complex preparation, limited drug loading capacity 2. Potential immunogenicity and biocompatibility issues	A protein gene therapy vector targeting TDP-43 was designed with high affinity to 14–3-3θ, effectively alleviating functional deficits and neurodegeneration in TDP-43 mutant ALS/FTD mouse models [47]
	Exosomes	1. Natural carriers with good immunogenicity and biocompatibility 2. Strong cell targeting and high stability in vivo	1. Difficult production and purification 2. Complexity of components and functions, which may lead to various adverse effects	Extracellular vesicles derived from stem cells targeting the optimization of the tryptophan-melatonin pathway in cells can be utilized for treating early-stage ALS [49]