药学学报

Drug name	Condition	Developer	Type	Target	Delivery method	Phase	Year approved or clinicaltrials.gov ID
Nusinersen (Spinraza)	SMA	Ionis/Biogen	ASOs	SMN2	IT	Approved (FDA)	2016
Tofersen (Qalsody)	ALS	Ionis/Biogen	ASOs	SOD1	IT	Approved (FDA)	2023
Ulefnersen (ION363)	ALS	Ionis	ASOs	FUS	IT	Ⅲ	NCT04768972
Zilganersen (ION373)	Alexander disease	Ionis	ASOs	GFAP	IT	Ⅲ	NCT04849741
Tominersen (IONIS-HTTRx)	HD	Ionis/Roche	ASOs	HTT	IT	Ⅱ	NCT05868551, NCT04000594
IONIS-MAPTRx (BIIB080)	AD	Ionis/Biogen	ASOs	MAPT	IT	Ⅱ	NCT03186989, NCT02835117
AMT-130	HD	UniQure Biopharma	Gene therapy (AAV vector)	HTT	Intra-striatal	Ⅰ/Ⅱ	NCT04120493
WVE-003	HD	Wave Life Sciences	siRNA	HTT	IT	Ⅰ/Ⅱ	NCT05032196
ALN-APP	Early-onset AD (EOAD)	Alnylam/Regeneron	siRNA	APP	IT	Ⅰ/Ⅱ	NCT05231785
STK-001	Dravet syndrome (DS)	Stoke Therapeutics	siRNA	SCN1A	IT	Ⅱ	NCT04740476
ION859 (BIIB094)	PD	Ionis/Biogen	ASOs	LRRK2	IT	Ⅰ/Ⅱ	NCT03976349
ION582	Angelman syndrome (AS)	Ionis/Biogen	ASOs	UBE3A-ATS	IT	Ⅰ/Ⅱ	NCT05127226
ION464 (BIIB101)	Multiple system atrophy (MSA)	Ionis	ASOs	SNCA	IT	Ⅰ/Ⅱ	NCT04165486

Drug name

Condition

Developer

Type

Target

Delivery method

Phase

Year approved or clinicaltrials.gov ID

Nusinersen
(Spinraza)

SMA

Ionis/Biogen

ASOs

SMN2

Approved
(FDA)

2016

Tofersen
(Qalsody)

ALS

Ionis/Biogen

ASOs

SOD1

Approved
(FDA)

2023

Ulefnersen
(ION363)

ALS

Ionis

ASOs

FUS

Ⅲ

NCT04768972

Zilganersen
(ION373)

Alexander disease

Ionis

ASOs

GFAP

Ⅲ

NCT04849741

Tominersen
(IONIS-HTTRx)

Ionis/Roche

ASOs

HTT

Ⅱ

NCT05868551,
NCT04000594

IONIS-MAPTRx
(BIIB080)

Ionis/Biogen

ASOs

MAPT

Ⅱ

NCT03186989,
NCT02835117

AMT-130

UniQure Biopharma

Gene therapy
(AAV vector)

HTT

Intra-striatal

Ⅰ/Ⅱ

NCT04120493

WVE-003

Wave Life Sciences

siRNA

HTT

Ⅰ/Ⅱ

NCT05032196

ALN-APP

Early-onset AD
(EOAD)

Alnylam/Regeneron

siRNA

APP

Ⅰ/Ⅱ

NCT05231785

STK-001

Dravet syndrome (DS)

Stoke Therapeutics

siRNA

SCN1A

Ⅱ

NCT04740476

ION859
(BIIB094)

Ionis/Biogen

ASOs

LRRK2

Ⅰ/Ⅱ

NCT03976349

ION582

Angelman syndrome (AS)

Ionis/Biogen

ASOs

UBE3A-ATS

Ⅰ/Ⅱ

NCT05127226

ION464
(BIIB101)

Multiple system atrophy (MSA)

Ionis

ASOs

SNCA

Ⅰ/Ⅱ

NCT04165486

Drug name	Condition	Developer	Type	Target	Delivery method	Phase	Year approved or clinicaltrials.gov ID
Nusinersen (Spinraza)	SMA	Ionis/Biogen	ASOs	SMN2	IT	Approved (FDA)	2016
Tofersen (Qalsody)	ALS	Ionis/Biogen	ASOs	SOD1	IT	Approved (FDA)	2023
Ulefnersen (ION363)	ALS	Ionis	ASOs	FUS	IT	Ⅲ	NCT04768972
Zilganersen (ION373)	Alexander disease	Ionis	ASOs	GFAP	IT	Ⅲ	NCT04849741
Tominersen (IONIS-HTTRx)	HD	Ionis/Roche	ASOs	HTT	IT	Ⅱ	NCT05868551, NCT04000594
IONIS-MAPTRx (BIIB080)	AD	Ionis/Biogen	ASOs	MAPT	IT	Ⅱ	NCT03186989, NCT02835117
AMT-130	HD	UniQure Biopharma	Gene therapy (AAV vector)	HTT	Intra-striatal	Ⅰ/Ⅱ	NCT04120493
WVE-003	HD	Wave Life Sciences	siRNA	HTT	IT	Ⅰ/Ⅱ	NCT05032196
ALN-APP	Early-onset AD (EOAD)	Alnylam/Regeneron	siRNA	APP	IT	Ⅰ/Ⅱ	NCT05231785
STK-001	Dravet syndrome (DS)	Stoke Therapeutics	siRNA	SCN1A	IT	Ⅱ	NCT04740476
ION859 (BIIB094)	PD	Ionis/Biogen	ASOs	LRRK2	IT	Ⅰ/Ⅱ	NCT03976349
ION582	Angelman syndrome (AS)	Ionis/Biogen	ASOs	UBE3A-ATS	IT	Ⅰ/Ⅱ	NCT05127226
ION464 (BIIB101)	Multiple system atrophy (MSA)	Ionis	ASOs	SNCA	IT	Ⅰ/Ⅱ	NCT04165486

Drug name

Condition

Developer

Type

Target

Delivery method

Phase

Year approved or clinicaltrials.gov ID

Nusinersen
(Spinraza)

SMA

Ionis/Biogen

ASOs

SMN2

Approved
(FDA)

2016

Tofersen
(Qalsody)

ALS

Ionis/Biogen

ASOs

SOD1

Approved
(FDA)

2023

Ulefnersen
(ION363)

ALS

Ionis

ASOs

FUS

Ⅲ

NCT04768972

Zilganersen
(ION373)

Alexander disease

Ionis

ASOs

GFAP

Ⅲ

NCT04849741

Tominersen
(IONIS-HTTRx)

Ionis/Roche

ASOs

HTT

Ⅱ

NCT05868551,
NCT04000594

IONIS-MAPTRx
(BIIB080)

Ionis/Biogen

ASOs

MAPT

Ⅱ

NCT03186989,
NCT02835117

AMT-130

UniQure Biopharma

Gene therapy
(AAV vector)

HTT

Intra-striatal

Ⅰ/Ⅱ

NCT04120493

WVE-003

Wave Life Sciences

siRNA

HTT

Ⅰ/Ⅱ

NCT05032196

ALN-APP

Early-onset AD
(EOAD)

Alnylam/Regeneron

siRNA

APP

Ⅰ/Ⅱ

NCT05231785

STK-001

Dravet syndrome (DS)

Stoke Therapeutics

siRNA

SCN1A

Ⅱ

NCT04740476

ION859
(BIIB094)

Ionis/Biogen

ASOs

LRRK2

Ⅰ/Ⅱ

NCT03976349

ION582

Angelman syndrome (AS)

Ionis/Biogen

ASOs

UBE3A-ATS

Ⅰ/Ⅱ

NCT05127226

ION464
(BIIB101)

Multiple system atrophy (MSA)

Ionis

ASOs

SNCA

Ⅰ/Ⅱ

NCT04165486

Type	Composition	Drug type	Mechanism	Disease	Target	Administration	Result	Ref.
Nucleic acid drug conjugation	DNA/RNA heteroduplex with cholesterol conjugation at the 5′ end of RNA strand	ASOs	Cholesterol-mediated transcytosis (cholesterol)	CNS-related conditions	Malat1	Ⅳ	Knockdown of Malat1 RNA in neurons, astrocytes, and microglial cells by up to 90%	[123]
HA2, ApoE-derived peptides, conjugated to phosphorodiamidate morpholino oligomer (PMO) antisense oligonucleotides	ASOs	RMT (ApoE peptides)	SMA	SMN2	Ⅳ	Improved exon-7 inclusion activity in the CNS, increased pharmacological efficacy and uptake in neuronal cells	[124]
Folate (FA)-conjugated RNA, anti-miR-21 LNA	siRNA	RMT (FA)	GBM	miRNA-21	Ⅳ	Effective knock down of miR-21 in glioblastoma rescue of tumor suppressor genes (PTEN, PDCD4), induced apoptosis	[125]
2′-O-Hexadecyl (C₁₆) conjugated siRNA	siRNA	Bypass BBB (IT or ICV)	AD	APP & SOD1	IT or ICV	Effective and durable gene silencing in CNS and lungs, up to 90% knockdown in animal models	[126]
Lipid-based nanoparticles	Lipid mixture (DODAP, DSPC, cholesterol, PEG-ceramide) encapsulating siRNAs with RVG-9r peptide	siRNA	RMT (TFR) (RVG)	Machado-Joseph disease (MJD)	ATXN3	Ⅳ	Significant reduction in mutant ataxin-3, reduced neuropathology, improved motor behavior in mouse models	[55]
Hyaluronic acid (HA), Dlin-MC3-DMA, DSPC, cholesterol, PEGylated lipids, siPLK1	siRNA	Bypass BBB (CDE)	GBM	PLK1	IC	80% Reduction in PLK1 mRNA levels, increased cell death in glioma cells, prolonged survival in GBM-bearing mice	[85]
PLGA, DPPC, Liss Rhod PE, DSPE-PEG, MVL5	siRNA	Open BBB (MB-FUS)	Glioma, medulloblastoma	SMO	Ⅳ	10-Fold increase in siRNA delivery, significant SMO knockdown, tumor reduction, and cancer cell apoptosis	[88]
DOPE, DOTAP, DSPE-PEG-COOH, Tf, penetratin, and chitosan-pDNA complex	pDNA	RMT & AMT (TFR) Tf & cpp	Neurodegenerative diseases	Neurons in the brain	Ⅳ	Efficient transfection of brain neurons and significant accumulation in the brain	[106]
SPIONs, DOPA-PLys-PEG-RVG, Flu	CRISPR/Cas9 fluvastatin	RMT (TFR) (RVG)	AD	BACE1	Ⅳ	Significantly improved cognition in AD mice, reduced amyloid-β plaques, efficient gene editing and synergistic effects	[110]
PEI-based hydrogel core, DOTAP, cholesterol, DSPE-PEG2000-MAL, iRGD-conjugation	CRISPR/Cas9	RMT & open BBB (iRGDs & LEX)	Brain tumour	PLK1	Ⅳ	Efficient PLK1 gene knockout, significant tumor inhibition, prolonged survival in mouse models	[98]
DSPE-PEG, EPC cholesterol, PEI, MAN, TAT peptide	miRNA	CMT (Glut1) & AMT (MAN ligand & TAT peptide)	AD	APP, BACE1	Ⅳ	Reduced Aβ and tau levels, improved cognitive function in mouse models	[127]
Ionizable lipid, cholesterol, phospholipid, PEG-lipid	siRNA	Bypass BBB (local intracisternal injection, IC)	AD	PU.1	IC	Successfully reduced neuroinflammation and decreased PU.1 levels in microglia	[128]
DPPC, DSPE-PEG2000, DSPE-COOH-PEG2000, DC-cholesterol	shRNA	Open BBB (FUS-MB)	Glioma	Birc5 gene, CD13	Ⅳ	Tumor growth was significantly reduced, with increased apoptosis and extended survival observed in glioma rats treated with FUS-assisted MB-shBirc5-lipo-NGR	[129]
DOPA, DMPC, ApoE3	siRNA	RMT (LDLR) (ApoE3)	GBM	ATF5	Ⅳ	Effective siRNA delivery, increased apoptosis inhibited tumor growth	[130]
DPPC, DOTAP, cholesterol	mRNA	Bypass BBB (nose to brain)	Neurological disorders	Brain neurons	IN	Significant mRNA transfection and protein expression in brain regions like cortex, striatum, midbrain; minimal systemic exposure	[131]
DPH/TPGAS NPs (composed of two amphiphilic polymers, D-PEG-Phis and T-PA-G-SA	siRNA	RMT (CTGF receptors) (DAG peptide)	AD	NF-κB, Aβ, tau protein	Ⅳ	Reduced Aβ and tau, improved memory and cognition in AD mouse models	[52]
PNP-based nanoparticles	PBAE, PEGylated	mRNA or CRISPR/Cas9 with sgRNA	Open BBB (FUS-MB)	Brain tumors, neurodegenerative diseases	/	Ⅳ	Effective nucleic acid delivery and genome editing in astrocytes and neurons, precise targeting of the FUS-treated brain region	[89]
Galactose-modified PEG-b-P(Gu), siRNA, fluorinated polymer	siRNA	CMT (Glut1)	AD	BACE1	Ⅳ	Reduced Aβ plaques, improved cognitive function, and reduced tau protein phosphorylation in AD mouse models	[119]
DGL conjugated with PEG and LIMK2 NoLS peptide	pDNA	AMT (LIMK2 NoLS peptide)	Glioma	ING4 gene (tumor suppressor)	Ⅳ	Enhanced BBB penetration, increased gene expression in tumor cells, increased apoptosis, extended survival	[122]
PEG-modified DGL, T7 peptide, Tet1 peptide	siRNA and D-peptide (Dp)	RMT(TFR) (T7 peptide & Tet1 peptide)	AD	BACE1, Aβ, p-tau	Ⅳ	Reduced β-amyloid plaques and tau protein phosphorylation; improved cognitive function in AD mice	[132]
PEGylated dendrigraft poly-L-lysines (DGL)	Non-coding RNA plasmid	RMT (RVG29 peptide)	AD	BACE1, tau protein	Ⅳ	Reduced Aβ plaques and tau fibrils, improved cognitive function, reduced neurodegeneration in AD mice	[133]
Glutathione-sensitive polymer, agiopep-2, Cas9/sgRNA	CRISPR/Cas9 complex	RMT(LRP) (Angiopep-2)	GBM	PLK1	Ⅳ	High PLK1 gene editing efficiency (up to 38.1%) in brain tumor, extended survival in mice (68 vs 24 days)	[114]
PEGylated DGL modified with Aleuria aurantia lectin (AAL) and KLVFF peptides	siRNA and rapamycin	Bypass BBB (nose to brain)	AD	BACE1, Aβ	IN	Reduced Aβ deposition, improved cognition, and induced autophagy in AD mice	[134]
Disulfide-crosslinked polymer, Angiopep-2	siRNA	RMT (LRP) (Angiopep-2)	GBM	PLK1	Ⅳ	Efficient BBB penetration, high tumor targeting, significant inhibition of glioblastoma growth, prolonged survival in GBM-bearing mice, minimal off-target effects	[135]
Transferrin-coated chimeric polymersomes (CPs)	siRNA	RMT (TFR) (TF)	Brain metastatic breast cancer	PLK1	Ⅳ	Effective tumor inhibition, enhanced survival, and specific RNA interference targeting metastatic brain tumors with transferrin-coated polymersomes	[103]
Exosomes derived from dendritic cells, modified with Lamp2b-RVG peptide	siRNA	RMT (nAChR) (RVG)	AD	BACE1	Ⅳ	Specific silencing of target genes (BACE1), reduction in mRNA and protein levels, reduced β-amyloid accumulation	[136]
	Exosomes derived from HEK293T cells, functionalized with an anti-EGFR single-chain variable fragment (scFv) antibody	siRNA	RMT (EGFR) (scFv)	NSCLC	LPCAT1	Ⅳ	Effectively delivered siRNA across the BBB to brain metastasis, showing significant antitumor efficacy with minimal toxicity	[137]
EV-based nanoparticles	Exosomes derived from hepatocytes, modified with LAMP2b-RVG29	siRNA	RMT (nAChR) (RVG29)	HD	HTT	Ⅳ	Reduction of mHTT protein and aggregates, improved motor function, and reduced neuropathology	[138]
Exosomes derived from serum of MCAO rats after electroacupuncture treatment	miRNA	Bypass BBB (IC)	Ischemic stroke	HIF-1α, VEGF, Notch1	IC	Decreased infarct volume, reduced pathological damage, increased angiogenic factor expression	[139]
Exosomes derived from HEK293 cell, decorated with T7-peptide	ASOs	RMT (TFR) (T7-peptide)	GBM	Anti-miR-21	Ⅳ	Significant reduction in tumor size, enhanced delivery to brain, higher uptake by glioblastoma cells	[140]
Macrophage-derived exosomes functionalized with panobinostat and PPM1D-siRNA	siRNA	RMT (cyclic RGD peptide, cRGD)	Gliomas	PPM1D	Ⅳ	Inhibited DIPG tumor growth, increased apoptosis, prolonged survival in DIPG-bearing mice	[141]

Type

Composition

Drug type

Mechanism

Disease

Target

Administration

Result

Ref.

Nucleic acid drug conjugation

DNA/RNA heteroduplex with cholesterol conjugation at the 5′ end of RNA strand

ASOs

Cholesterol-mediated transcytosis
(cholesterol)

CNS-related conditions

Malat1

Ⅳ

Knockdown of Malat1 RNA in neurons, astrocytes, and microglial cells by up to 90%

[123]

HA2, ApoE-derived peptides, conjugated to phosphorodiamidate morpholino oligomer (PMO) antisense oligonucleotides

ASOs

RMT (ApoE peptides)

SMA

SMN2

Ⅳ

Improved exon-7 inclusion activity in the CNS, increased pharmacological efficacy and uptake in neuronal cells

[124]

Folate (FA)-conjugated RNA, anti-miR-21 LNA

siRNA

RMT (FA)

GBM

miRNA-21

Ⅳ

Effective knock down of miR-21 in glioblastoma rescue of tumor suppressor genes (PTEN, PDCD4), induced apoptosis

[125]

2′-O-Hexadecyl (C₁₆) conjugated siRNA

siRNA

Bypass BBB (IT or ICV)

APP & SOD1

IT or ICV

Effective and durable gene silencing in CNS and lungs, up to 90% knockdown in animal models

[126]

Lipid-based nanoparticles

Lipid mixture (DODAP, DSPC, cholesterol, PEG-ceramide) encapsulating siRNAs with RVG-9r peptide

siRNA

RMT (TFR)
(RVG)

Machado-Joseph disease (MJD)

ATXN3

Ⅳ

Significant reduction in mutant ataxin-3, reduced neuropathology, improved motor behavior in mouse models

[55]

Hyaluronic acid (HA), Dlin-MC3-DMA, DSPC, cholesterol, PEGylated lipids, siPLK1

siRNA

Bypass BBB (CDE)

GBM

PLK1

80% Reduction in PLK1 mRNA levels, increased cell death in glioma cells, prolonged survival in GBM-bearing mice

[85]

PLGA, DPPC, Liss Rhod PE, DSPE-PEG, MVL5

siRNA

Open BBB
(MB-FUS)

Glioma, medulloblastoma

SMO

Ⅳ

10-Fold increase in siRNA delivery, significant SMO knockdown, tumor reduction, and cancer cell apoptosis

[88]

DOPE, DOTAP, DSPE-PEG-COOH, Tf, penetratin, and chitosan-pDNA complex

pDNA

RMT & AMT (TFR)
Tf & cpp

Neurodegenerative diseases

Neurons in the brain

Ⅳ

Efficient transfection of brain neurons and significant accumulation in the brain

[106]

SPIONs, DOPA-PLys-PEG-RVG, Flu

CRISPR/Cas9 fluvastatin

RMT (TFR)
(RVG)

BACE1

Ⅳ

Significantly improved cognition in AD mice, reduced amyloid-β plaques, efficient gene editing and synergistic effects

[110]

PEI-based hydrogel core, DOTAP, cholesterol, DSPE-PEG2000-MAL, iRGD-conjugation

CRISPR/Cas9

RMT & open BBB
(iRGDs & LEX)

Brain tumour

PLK1

Ⅳ

Efficient PLK1 gene knockout, significant tumor inhibition, prolonged survival in mouse models

[98]

DSPE-PEG, EPC cholesterol, PEI, MAN, TAT peptide

miRNA

CMT (Glut1) & AMT (MAN ligand & TAT peptide)

APP, BACE1

Ⅳ

Reduced Aβ and tau levels, improved cognitive function in mouse models

[127]

Ionizable lipid, cholesterol, phospholipid, PEG-lipid

siRNA

Bypass BBB
(local intracisternal injection, IC)

PU.1

Successfully reduced neuroinflammation and decreased PU.1 levels in microglia

[128]

DPPC, DSPE-PEG2000, DSPE-COOH-PEG2000, DC-cholesterol

shRNA

Open BBB
(FUS-MB)

Glioma

Birc5 gene, CD13

Ⅳ

Tumor growth was significantly reduced, with increased apoptosis and extended survival observed in glioma rats treated with FUS-assisted MB-shBirc5-lipo-NGR

[129]

DOPA, DMPC, ApoE3

siRNA

RMT (LDLR)
(ApoE3)

GBM

ATF5

Ⅳ

Effective siRNA delivery, increased apoptosis inhibited tumor growth

[130]

DPPC, DOTAP, cholesterol

mRNA

Bypass BBB
(nose to brain)

Neurological disorders

Brain neurons

Significant mRNA transfection and protein expression in brain regions like cortex, striatum, midbrain; minimal systemic exposure

[131]

DPH/TPGAS NPs (composed of two amphiphilic polymers, D-PEG-Phis and T-PA-G-SA

siRNA

RMT (CTGF receptors)
(DAG peptide)

NF-κB, Aβ, tau protein

Ⅳ

Reduced Aβ and tau, improved memory and cognition in AD mouse models

[52]

PNP-based nanoparticles

PBAE, PEGylated

mRNA or CRISPR/Cas9 with sgRNA

Open BBB
(FUS-MB)

Brain tumors, neurodegenerative diseases

Ⅳ

Effective nucleic acid delivery and genome editing in astrocytes and neurons, precise targeting of the FUS-treated brain region

[89]

Galactose-modified PEG-b-P(Gu), siRNA, fluorinated polymer

siRNA

CMT
(Glut1)

BACE1

Ⅳ

Reduced Aβ plaques, improved cognitive function, and reduced tau protein phosphorylation in AD mouse models

[119]

DGL conjugated with PEG and LIMK2 NoLS peptide

pDNA

AMT
(LIMK2 NoLS peptide)

Glioma

ING4 gene (tumor suppressor)

Ⅳ

Enhanced BBB penetration, increased gene expression in tumor cells, increased apoptosis, extended survival

[122]

PEG-modified DGL, T7 peptide, Tet1 peptide

siRNA and D-peptide (Dp)

RMT(TFR)
(T7 peptide & Tet1 peptide)

BACE1, Aβ, p-tau

Ⅳ

Reduced β-amyloid plaques and tau protein phosphorylation; improved cognitive function in AD mice

[132]

PEGylated dendrigraft poly-L-lysines (DGL)

Non-coding RNA plasmid

RMT
(RVG29 peptide)

BACE1, tau protein

Ⅳ

Reduced Aβ plaques and tau fibrils, improved cognitive function, reduced neurodegeneration in AD mice

[133]

Glutathione-sensitive polymer, agiopep-2, Cas9/sgRNA

CRISPR/Cas9 complex

RMT(LRP)
(Angiopep-2)

GBM

PLK1

Ⅳ

High PLK1 gene editing efficiency (up to 38.1%) in brain tumor, extended survival in mice (68 vs 24 days)

[114]

PEGylated DGL modified with Aleuria aurantia lectin (AAL) and KLVFF peptides

siRNA and rapamycin

Bypass BBB
(nose to brain)

BACE1, Aβ

Reduced Aβ deposition, improved cognition, and induced autophagy in AD mice

[134]

Disulfide-crosslinked polymer, Angiopep-2

siRNA

RMT (LRP)
(Angiopep-2)

GBM

PLK1

Ⅳ

Efficient BBB penetration, high tumor targeting, significant inhibition of glioblastoma growth, prolonged survival in GBM-bearing mice, minimal off-target effects

[135]

Transferrin-coated chimeric polymersomes (CPs)

siRNA

RMT (TFR)
(TF)

Brain metastatic breast cancer

PLK1

Ⅳ

Effective tumor inhibition, enhanced survival, and specific RNA interference targeting metastatic brain tumors with transferrin-coated polymersomes

[103]

Exosomes derived from dendritic cells, modified with Lamp2b-RVG peptide

siRNA

RMT (nAChR)
(RVG)

BACE1

Ⅳ

Specific silencing of target genes (BACE1), reduction in mRNA and protein levels, reduced β-amyloid accumulation

[136]

Exosomes derived from HEK293T cells, functionalized with an anti-EGFR single-chain variable fragment (scFv) antibody

siRNA

RMT (EGFR)
(scFv)

NSCLC

LPCAT1

Ⅳ

Effectively delivered siRNA across the BBB to brain metastasis, showing significant antitumor efficacy with minimal toxicity

[137]

EV-based nanoparticles

Exosomes derived from hepatocytes, modified with LAMP2b-RVG29

siRNA

RMT (nAChR)
(RVG29)

HTT

Ⅳ

Reduction of mHTT protein and aggregates, improved motor function, and reduced neuropathology

[138]

Exosomes derived from serum of MCAO rats after electroacupuncture treatment

miRNA

Bypass BBB
(IC)

Ischemic stroke

HIF-1α, VEGF, Notch1

Decreased infarct volume, reduced pathological damage, increased angiogenic factor expression

[139]

Exosomes derived from HEK293 cell, decorated with T7-peptide

ASOs

RMT (TFR)
(T7-peptide)

GBM

Anti-miR-21

Ⅳ

Significant reduction in tumor size, enhanced delivery to brain, higher uptake by glioblastoma cells

[140]

Macrophage-derived exosomes functionalized with panobinostat and PPM1D-siRNA

siRNA

RMT
(cyclic RGD peptide, cRGD)

Gliomas

PPM1D

Ⅳ

Inhibited DIPG tumor growth, increased apoptosis, prolonged survival in DIPG-bearing mice

[141]

Type	Composition	Drug type	Mechanism	Disease	Target	Administration	Result	Ref.
Nucleic acid drug conjugation	DNA/RNA heteroduplex with cholesterol conjugation at the 5′ end of RNA strand	ASOs	Cholesterol-mediated transcytosis (cholesterol)	CNS-related conditions	Malat1	Ⅳ	Knockdown of Malat1 RNA in neurons, astrocytes, and microglial cells by up to 90%	[123]
HA2, ApoE-derived peptides, conjugated to phosphorodiamidate morpholino oligomer (PMO) antisense oligonucleotides	ASOs	RMT (ApoE peptides)	SMA	SMN2	Ⅳ	Improved exon-7 inclusion activity in the CNS, increased pharmacological efficacy and uptake in neuronal cells	[124]
Folate (FA)-conjugated RNA, anti-miR-21 LNA	siRNA	RMT (FA)	GBM	miRNA-21	Ⅳ	Effective knock down of miR-21 in glioblastoma rescue of tumor suppressor genes (PTEN, PDCD4), induced apoptosis	[125]
2′-O-Hexadecyl (C₁₆) conjugated siRNA	siRNA	Bypass BBB (IT or ICV)	AD	APP & SOD1	IT or ICV	Effective and durable gene silencing in CNS and lungs, up to 90% knockdown in animal models	[126]
Lipid-based nanoparticles	Lipid mixture (DODAP, DSPC, cholesterol, PEG-ceramide) encapsulating siRNAs with RVG-9r peptide	siRNA	RMT (TFR) (RVG)	Machado-Joseph disease (MJD)	ATXN3	Ⅳ	Significant reduction in mutant ataxin-3, reduced neuropathology, improved motor behavior in mouse models	[55]
Hyaluronic acid (HA), Dlin-MC3-DMA, DSPC, cholesterol, PEGylated lipids, siPLK1	siRNA	Bypass BBB (CDE)	GBM	PLK1	IC	80% Reduction in PLK1 mRNA levels, increased cell death in glioma cells, prolonged survival in GBM-bearing mice	[85]
PLGA, DPPC, Liss Rhod PE, DSPE-PEG, MVL5	siRNA	Open BBB (MB-FUS)	Glioma, medulloblastoma	SMO	Ⅳ	10-Fold increase in siRNA delivery, significant SMO knockdown, tumor reduction, and cancer cell apoptosis	[88]
DOPE, DOTAP, DSPE-PEG-COOH, Tf, penetratin, and chitosan-pDNA complex	pDNA	RMT & AMT (TFR) Tf & cpp	Neurodegenerative diseases	Neurons in the brain	Ⅳ	Efficient transfection of brain neurons and significant accumulation in the brain	[106]
SPIONs, DOPA-PLys-PEG-RVG, Flu	CRISPR/Cas9 fluvastatin	RMT (TFR) (RVG)	AD	BACE1	Ⅳ	Significantly improved cognition in AD mice, reduced amyloid-β plaques, efficient gene editing and synergistic effects	[110]
PEI-based hydrogel core, DOTAP, cholesterol, DSPE-PEG2000-MAL, iRGD-conjugation	CRISPR/Cas9	RMT & open BBB (iRGDs & LEX)	Brain tumour	PLK1	Ⅳ	Efficient PLK1 gene knockout, significant tumor inhibition, prolonged survival in mouse models	[98]
DSPE-PEG, EPC cholesterol, PEI, MAN, TAT peptide	miRNA	CMT (Glut1) & AMT (MAN ligand & TAT peptide)	AD	APP, BACE1	Ⅳ	Reduced Aβ and tau levels, improved cognitive function in mouse models	[127]
Ionizable lipid, cholesterol, phospholipid, PEG-lipid	siRNA	Bypass BBB (local intracisternal injection, IC)	AD	PU.1	IC	Successfully reduced neuroinflammation and decreased PU.1 levels in microglia	[128]
DPPC, DSPE-PEG2000, DSPE-COOH-PEG2000, DC-cholesterol	shRNA	Open BBB (FUS-MB)	Glioma	Birc5 gene, CD13	Ⅳ	Tumor growth was significantly reduced, with increased apoptosis and extended survival observed in glioma rats treated with FUS-assisted MB-shBirc5-lipo-NGR	[129]
DOPA, DMPC, ApoE3	siRNA	RMT (LDLR) (ApoE3)	GBM	ATF5	Ⅳ	Effective siRNA delivery, increased apoptosis inhibited tumor growth	[130]
DPPC, DOTAP, cholesterol	mRNA	Bypass BBB (nose to brain)	Neurological disorders	Brain neurons	IN	Significant mRNA transfection and protein expression in brain regions like cortex, striatum, midbrain; minimal systemic exposure	[131]
DPH/TPGAS NPs (composed of two amphiphilic polymers, D-PEG-Phis and T-PA-G-SA	siRNA	RMT (CTGF receptors) (DAG peptide)	AD	NF-κB, Aβ, tau protein	Ⅳ	Reduced Aβ and tau, improved memory and cognition in AD mouse models	[52]
PNP-based nanoparticles	PBAE, PEGylated	mRNA or CRISPR/Cas9 with sgRNA	Open BBB (FUS-MB)	Brain tumors, neurodegenerative diseases	/	Ⅳ	Effective nucleic acid delivery and genome editing in astrocytes and neurons, precise targeting of the FUS-treated brain region	[89]
Galactose-modified PEG-b-P(Gu), siRNA, fluorinated polymer	siRNA	CMT (Glut1)	AD	BACE1	Ⅳ	Reduced Aβ plaques, improved cognitive function, and reduced tau protein phosphorylation in AD mouse models	[119]
DGL conjugated with PEG and LIMK2 NoLS peptide	pDNA	AMT (LIMK2 NoLS peptide)	Glioma	ING4 gene (tumor suppressor)	Ⅳ	Enhanced BBB penetration, increased gene expression in tumor cells, increased apoptosis, extended survival	[122]
PEG-modified DGL, T7 peptide, Tet1 peptide	siRNA and D-peptide (Dp)	RMT(TFR) (T7 peptide & Tet1 peptide)	AD	BACE1, Aβ, p-tau	Ⅳ	Reduced β-amyloid plaques and tau protein phosphorylation; improved cognitive function in AD mice	[132]
PEGylated dendrigraft poly-L-lysines (DGL)	Non-coding RNA plasmid	RMT (RVG29 peptide)	AD	BACE1, tau protein	Ⅳ	Reduced Aβ plaques and tau fibrils, improved cognitive function, reduced neurodegeneration in AD mice	[133]
Glutathione-sensitive polymer, agiopep-2, Cas9/sgRNA	CRISPR/Cas9 complex	RMT(LRP) (Angiopep-2)	GBM	PLK1	Ⅳ	High PLK1 gene editing efficiency (up to 38.1%) in brain tumor, extended survival in mice (68 vs 24 days)	[114]
PEGylated DGL modified with Aleuria aurantia lectin (AAL) and KLVFF peptides	siRNA and rapamycin	Bypass BBB (nose to brain)	AD	BACE1, Aβ	IN	Reduced Aβ deposition, improved cognition, and induced autophagy in AD mice	[134]
Disulfide-crosslinked polymer, Angiopep-2	siRNA	RMT (LRP) (Angiopep-2)	GBM	PLK1	Ⅳ	Efficient BBB penetration, high tumor targeting, significant inhibition of glioblastoma growth, prolonged survival in GBM-bearing mice, minimal off-target effects	[135]
Transferrin-coated chimeric polymersomes (CPs)	siRNA	RMT (TFR) (TF)	Brain metastatic breast cancer	PLK1	Ⅳ	Effective tumor inhibition, enhanced survival, and specific RNA interference targeting metastatic brain tumors with transferrin-coated polymersomes	[103]
Exosomes derived from dendritic cells, modified with Lamp2b-RVG peptide	siRNA	RMT (nAChR) (RVG)	AD	BACE1	Ⅳ	Specific silencing of target genes (BACE1), reduction in mRNA and protein levels, reduced β-amyloid accumulation	[136]
	Exosomes derived from HEK293T cells, functionalized with an anti-EGFR single-chain variable fragment (scFv) antibody	siRNA	RMT (EGFR) (scFv)	NSCLC	LPCAT1	Ⅳ	Effectively delivered siRNA across the BBB to brain metastasis, showing significant antitumor efficacy with minimal toxicity	[137]
EV-based nanoparticles	Exosomes derived from hepatocytes, modified with LAMP2b-RVG29	siRNA	RMT (nAChR) (RVG29)	HD	HTT	Ⅳ	Reduction of mHTT protein and aggregates, improved motor function, and reduced neuropathology	[138]
Exosomes derived from serum of MCAO rats after electroacupuncture treatment	miRNA	Bypass BBB (IC)	Ischemic stroke	HIF-1α, VEGF, Notch1	IC	Decreased infarct volume, reduced pathological damage, increased angiogenic factor expression	[139]
Exosomes derived from HEK293 cell, decorated with T7-peptide	ASOs	RMT (TFR) (T7-peptide)	GBM	Anti-miR-21	Ⅳ	Significant reduction in tumor size, enhanced delivery to brain, higher uptake by glioblastoma cells	[140]
Macrophage-derived exosomes functionalized with panobinostat and PPM1D-siRNA	siRNA	RMT (cyclic RGD peptide, cRGD)	Gliomas	PPM1D	Ⅳ	Inhibited DIPG tumor growth, increased apoptosis, prolonged survival in DIPG-bearing mice	[141]

Type

Composition

Drug type

Mechanism

Disease

Target

Administration

Result

Ref.

Nucleic acid drug conjugation

DNA/RNA heteroduplex with cholesterol conjugation at the 5′ end of RNA strand

ASOs

Cholesterol-mediated transcytosis
(cholesterol)

CNS-related conditions

Malat1

Ⅳ

Knockdown of Malat1 RNA in neurons, astrocytes, and microglial cells by up to 90%

[123]

HA2, ApoE-derived peptides, conjugated to phosphorodiamidate morpholino oligomer (PMO) antisense oligonucleotides

ASOs

RMT (ApoE peptides)

SMA

SMN2

Ⅳ

Improved exon-7 inclusion activity in the CNS, increased pharmacological efficacy and uptake in neuronal cells

[124]

Folate (FA)-conjugated RNA, anti-miR-21 LNA

siRNA

RMT (FA)

GBM

miRNA-21

Ⅳ

Effective knock down of miR-21 in glioblastoma rescue of tumor suppressor genes (PTEN, PDCD4), induced apoptosis

[125]

2′-O-Hexadecyl (C₁₆) conjugated siRNA

siRNA

Bypass BBB (IT or ICV)

APP & SOD1

IT or ICV

Effective and durable gene silencing in CNS and lungs, up to 90% knockdown in animal models

[126]

Lipid-based nanoparticles

Lipid mixture (DODAP, DSPC, cholesterol, PEG-ceramide) encapsulating siRNAs with RVG-9r peptide

siRNA

RMT (TFR)
(RVG)

Machado-Joseph disease (MJD)

ATXN3

Ⅳ

Significant reduction in mutant ataxin-3, reduced neuropathology, improved motor behavior in mouse models

[55]

Hyaluronic acid (HA), Dlin-MC3-DMA, DSPC, cholesterol, PEGylated lipids, siPLK1

siRNA

Bypass BBB (CDE)

GBM

PLK1

80% Reduction in PLK1 mRNA levels, increased cell death in glioma cells, prolonged survival in GBM-bearing mice

[85]

PLGA, DPPC, Liss Rhod PE, DSPE-PEG, MVL5

siRNA

Open BBB
(MB-FUS)

Glioma, medulloblastoma

SMO

Ⅳ

10-Fold increase in siRNA delivery, significant SMO knockdown, tumor reduction, and cancer cell apoptosis

[88]

DOPE, DOTAP, DSPE-PEG-COOH, Tf, penetratin, and chitosan-pDNA complex

pDNA

RMT & AMT (TFR)
Tf & cpp

Neurodegenerative diseases

Neurons in the brain

Ⅳ

Efficient transfection of brain neurons and significant accumulation in the brain

[106]

SPIONs, DOPA-PLys-PEG-RVG, Flu

CRISPR/Cas9 fluvastatin

RMT (TFR)
(RVG)

BACE1

Ⅳ

Significantly improved cognition in AD mice, reduced amyloid-β plaques, efficient gene editing and synergistic effects

[110]

PEI-based hydrogel core, DOTAP, cholesterol, DSPE-PEG2000-MAL, iRGD-conjugation

CRISPR/Cas9

RMT & open BBB
(iRGDs & LEX)

Brain tumour

PLK1

Ⅳ

Efficient PLK1 gene knockout, significant tumor inhibition, prolonged survival in mouse models

[98]

DSPE-PEG, EPC cholesterol, PEI, MAN, TAT peptide

miRNA

CMT (Glut1) & AMT (MAN ligand & TAT peptide)

APP, BACE1

Ⅳ

Reduced Aβ and tau levels, improved cognitive function in mouse models

[127]

Ionizable lipid, cholesterol, phospholipid, PEG-lipid

siRNA

Bypass BBB
(local intracisternal injection, IC)

PU.1

Successfully reduced neuroinflammation and decreased PU.1 levels in microglia

[128]

DPPC, DSPE-PEG2000, DSPE-COOH-PEG2000, DC-cholesterol

shRNA

Open BBB
(FUS-MB)

Glioma

Birc5 gene, CD13

Ⅳ

Tumor growth was significantly reduced, with increased apoptosis and extended survival observed in glioma rats treated with FUS-assisted MB-shBirc5-lipo-NGR

[129]

DOPA, DMPC, ApoE3

siRNA

RMT (LDLR)
(ApoE3)

GBM

ATF5

Ⅳ

Effective siRNA delivery, increased apoptosis inhibited tumor growth

[130]

DPPC, DOTAP, cholesterol

mRNA

Bypass BBB
(nose to brain)

Neurological disorders

Brain neurons

Significant mRNA transfection and protein expression in brain regions like cortex, striatum, midbrain; minimal systemic exposure

[131]

DPH/TPGAS NPs (composed of two amphiphilic polymers, D-PEG-Phis and T-PA-G-SA

siRNA

RMT (CTGF receptors)
(DAG peptide)

NF-κB, Aβ, tau protein

Ⅳ

Reduced Aβ and tau, improved memory and cognition in AD mouse models

[52]

PNP-based nanoparticles

PBAE, PEGylated

mRNA or CRISPR/Cas9 with sgRNA

Open BBB
(FUS-MB)

Brain tumors, neurodegenerative diseases

Ⅳ

Effective nucleic acid delivery and genome editing in astrocytes and neurons, precise targeting of the FUS-treated brain region

[89]

Galactose-modified PEG-b-P(Gu), siRNA, fluorinated polymer

siRNA

CMT
(Glut1)

BACE1

Ⅳ

Reduced Aβ plaques, improved cognitive function, and reduced tau protein phosphorylation in AD mouse models

[119]

DGL conjugated with PEG and LIMK2 NoLS peptide

pDNA

AMT
(LIMK2 NoLS peptide)

Glioma

ING4 gene (tumor suppressor)

Ⅳ

Enhanced BBB penetration, increased gene expression in tumor cells, increased apoptosis, extended survival

[122]

PEG-modified DGL, T7 peptide, Tet1 peptide

siRNA and D-peptide (Dp)

RMT(TFR)
(T7 peptide & Tet1 peptide)

BACE1, Aβ, p-tau

Ⅳ

Reduced β-amyloid plaques and tau protein phosphorylation; improved cognitive function in AD mice

[132]

PEGylated dendrigraft poly-L-lysines (DGL)

Non-coding RNA plasmid

RMT
(RVG29 peptide)

BACE1, tau protein

Ⅳ

Reduced Aβ plaques and tau fibrils, improved cognitive function, reduced neurodegeneration in AD mice

[133]

Glutathione-sensitive polymer, agiopep-2, Cas9/sgRNA

CRISPR/Cas9 complex

RMT(LRP)
(Angiopep-2)

GBM

PLK1

Ⅳ

High PLK1 gene editing efficiency (up to 38.1%) in brain tumor, extended survival in mice (68 vs 24 days)

[114]

PEGylated DGL modified with Aleuria aurantia lectin (AAL) and KLVFF peptides

siRNA and rapamycin

Bypass BBB
(nose to brain)

BACE1, Aβ

Reduced Aβ deposition, improved cognition, and induced autophagy in AD mice

[134]

Disulfide-crosslinked polymer, Angiopep-2

siRNA

RMT (LRP)
(Angiopep-2)

GBM

PLK1

Ⅳ

Efficient BBB penetration, high tumor targeting, significant inhibition of glioblastoma growth, prolonged survival in GBM-bearing mice, minimal off-target effects

[135]

Transferrin-coated chimeric polymersomes (CPs)

siRNA

RMT (TFR)
(TF)

Brain metastatic breast cancer

PLK1

Ⅳ

Effective tumor inhibition, enhanced survival, and specific RNA interference targeting metastatic brain tumors with transferrin-coated polymersomes

[103]

Exosomes derived from dendritic cells, modified with Lamp2b-RVG peptide

siRNA

RMT (nAChR)
(RVG)

BACE1

Ⅳ

Specific silencing of target genes (BACE1), reduction in mRNA and protein levels, reduced β-amyloid accumulation

[136]

Exosomes derived from HEK293T cells, functionalized with an anti-EGFR single-chain variable fragment (scFv) antibody

siRNA

RMT (EGFR)
(scFv)

NSCLC

LPCAT1

Ⅳ

Effectively delivered siRNA across the BBB to brain metastasis, showing significant antitumor efficacy with minimal toxicity

[137]

EV-based nanoparticles

Exosomes derived from hepatocytes, modified with LAMP2b-RVG29

siRNA

RMT (nAChR)
(RVG29)

HTT

Ⅳ

Reduction of mHTT protein and aggregates, improved motor function, and reduced neuropathology

[138]

Exosomes derived from serum of MCAO rats after electroacupuncture treatment

miRNA

Bypass BBB
(IC)

Ischemic stroke

HIF-1α, VEGF, Notch1

Decreased infarct volume, reduced pathological damage, increased angiogenic factor expression

[139]

Exosomes derived from HEK293 cell, decorated with T7-peptide

ASOs

RMT (TFR)
(T7-peptide)

GBM

Anti-miR-21

Ⅳ

Significant reduction in tumor size, enhanced delivery to brain, higher uptake by glioblastoma cells

[140]

Macrophage-derived exosomes functionalized with panobinostat and PPM1D-siRNA

siRNA

RMT
(cyclic RGD peptide, cRGD)

Gliomas

PPM1D

Ⅳ

Inhibited DIPG tumor growth, increased apoptosis, prolonged survival in DIPG-bearing mice

[141]

核酸药物的脑部递送研究进展

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施雯 , 黄宇坤 , 高小玲 ^*

药学学报 | 综述 2025,60(8): 2486-2509

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药学学报 |综述 2025 , 60 (8) : 2486 -2509

核酸药物的脑部递送研究进展

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施雯, 黄宇坤, 高小玲^*

作者信息

上海交通大学医学院, 上海 200025

通讯作者:

高小玲, Tel: 86-21-63846590-776580, E-mail: shellygao1@sjtu.edu.cn

作者简介:

作者贡献:

施雯负责撰写; 黄宇坤和高小玲负责设计与修改。

Advances in brain delivery of nucleic acid drugs

Wen SHI, Yu-kun HUANG, Xiao-ling GAO^*

Affiliations

School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China

出版时间: 2025-08-12 doi: 10.16438/j.0513-4870.2024-1196

文章导航

摘要

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中枢神经系统疾病是全球性的公共卫生问题, 随人口老龄化而加剧。核酸药物在该领域潜力巨大, 通过碱基互补配对原理靶向特定基因序列, 于蛋白质表达前进行干预, 具有靶向性高、安全性好且研发周期短的优势。然而, 血脑屏障的存在和中枢神经系统的病理复杂性限制了核酸药物的递送, 其疗效依赖于有效的递送系统。本文系统介绍了近年来用于治疗中枢神经系统疾病的核酸药物及其作用机制, 深入分析脑部递送障碍, 重点阐述了血脑屏障的生理结构、功能及可实现递送的途径及其相关机制。详述了利用病毒载体、核酸药物改造技术、脂质纳米颗粒、聚合物纳米颗粒和外泌体等突破血脑屏障的机制及其在核酸药物递送中的应用。本综述旨在汇总和分析当前核酸药物跨越血脑屏障、提高靶向性和稳定性的策略, 探讨提高递送效率的方法, 并为未来的研究和临床应用提供指导, 以期推动核酸药物在治疗中枢神经系统疾病方面的发展。

关键词

中枢神经系统疾病 / 血脑屏障 / 核酸药物 / 基因治疗 / 药物递送系统

Abstract

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Central nervous system (CNS) diseases are a global public health concern, exacerbated by the aging population. Nucleic acid drugs have shown great potential in this field. They target specific gene sequences through the principle of complementary base pairing, allowing intervention before protein expression. These drugs offer high specificity, good safety profiles, and relatively short development cycles. However, the presence of the blood-brain barrier (BBB) and the complexity of CNS pathologies restrict drug delivery, making effective delivery systems crucial for the therapeutic effect of nucleic acid drugs. This review introduces nucleic acid drugs developed for CNS disease treatment in recent years, along with their mechanisms of action. It analyzes the obstacles encountered in brain-targeted delivery of nucleic acid drugs, focusing on the physiological structure and function of the BBB, as well as potential pathways and associated mechanisms for achieving delivery. It details the mechanisms of utilizing viral vectors, nucleic acid drug modification techniques, lipid nanoparticles, polymeric nanoparticles, and exosomes to penetrate the blood-brain barrier, as well as their applications in the delivery of nucleic acid drugs. The aim of this review is to compile and analyze current strategies for enabling nucleic acid drugs to cross the BBB while improving targeting and stability. It explores methods to enhance delivery efficiency and provides guidance for future research and clinical application, ultimately promoting the development of nucleic acid therapeutics for CNS disease treatment.

Key words

central nervous system disease / blood-brain barrier / nucleic acid drug / genetic therapy / drug delivery system

引用本文

施雯, 黄宇坤, 高小玲. 核酸药物的脑部递送研究进展. 药学学报, 2025 , 60 (8) : 2486 -2509 . DOI: 10.16438/j.0513-4870.2024-1196

Wen SHI, Yu-kun HUANG, Xiao-ling GAO. Advances in brain delivery of nucleic acid drugs[J]. Acta Pharmaceutica Sinica, 2025 , 60 (8) : 2486 -2509 . DOI: 10.16438/j.0513-4870.2024-1196

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中枢神经系统(central nervous system, CNS) 包括大脑和脊髓, 是人体神经系统的核心部分。中枢神经系统疾病是全球健康的重大挑战, 包括阿尔茨海默病(Alzheimer's disease, AD)、帕金森病(Parkinson's disease, PD) 及亨廷顿病(Huntington's disease, HD) 等神经退行性疾病, 自闭症谱系障碍、Rett综合征等神经发育障碍, 以及脑卒中、脑肿瘤和多发性硬化症等^{[1, 2]}。这些疾病影响着全球数亿人口, 已成为全球第二大死亡原因, 其死亡率和致残率负担随人口老龄化加剧而持续上升^[3]。

传统药物治疗中枢神经系统疾病面临诸多挑战, 其中血脑屏障(blood-brain barrier, BBB) 是关键限制因素^[4]。BBB由脑毛细血管内皮细胞与神经血管单元中的周细胞、星形胶质细胞、神经元和小胶质细胞相互作用形成, 是一种选择性通透屏障。它在保护中枢神经系统免受毒素、病原体、炎症、损伤和疾病侵害等方面发挥着重要作用, 但同时也限制了大多数候选药物通过外周给药进入^{[5, 6]}。因此, 目前神经系统疾病的治疗药物有限, 可用的治疗方案少, 新药的批准率低^[7]。

在神经系统疾病的治疗中, 部分传统小分子药物因其较小的分子质量和较高的脂溶性, 能够有效穿透BBB, 通过调节神经递质、调控离子通道及提供神经保护等机制发挥作用。例如, 左旋多巴用于改善帕金森病症状^[8]、苯妥英钠用于癫痫发作^[9], 以及多奈哌齐用于阿尔茨海默病缓减症状^[10]等。但这类药物通常存在特异性弱、难以精准调控基因层面的病理机制等局限性。蛋白药物(如单克隆抗体和神经生长因子等) 具有较好的特异性, 通常通过特异性结合病理分子、调节信号通路并清除有害蛋白等方式实现治疗效果, 如单克隆抗体可通过清除特定的致病蛋白(如阿尔茨海默病中的β-淀粉样蛋白) 来减缓疾病进展^[11]。但蛋白类药物在临床应用中也面临着BBB穿透困难、生产成本高及潜在的免疫反应等挑战。

相比之下, 核酸药物在治疗中枢神经系统疾病中展现出显著优势^[12]。传统药物主要针对蛋白靶点, 仅覆盖已知致病靶点的15%, 而核酸药物通过干预基因转录和信使RNA (messenger RNA, mRNA) 环节, 理论上可靶向任何可转录基因序列, 极大扩展了潜在的治疗靶标^[13]。基于碱基配对的序列特异性设计, 核酸药物能够精准识别目标基因, 显著降低脱靶效应, 提高治疗安全性。此外, 核酸药物具有较高的生产效率, 一旦解决其递送和化学性质等关键问题, 仅需改变靶基因序列即可快速开发新药, 大幅缩短研发周期^{[14, 15]}。然而, 核酸药物需在胞内发挥作用, 其固有的高负电荷密度及体内核酸酶引起的不稳定性要求核酸药物的体内递送需依赖载体介导^[16]。因此, 载体的开发需要基于对体内生理屏障的深入研究, 包括循环中的降解清除与蛋白吸附、BBB、细胞摄取障碍、内吞体/溶酶体屏障, 以及药物控制释放机制等。目前, 聚合物纳米颗粒、脂质纳米颗粒、无机纳米颗粒和外泌体等工程载体已被广泛开发用于将核酸药物输送到大脑。通过调整载体的物理和化学特性, 引入官能团进行表面修饰, 可增强载体的稳定性、提高其对免疫监控的逃逸能力、优化器官靶向性和细胞定向性, 以及提升对微环境的敏感性等。

本文介绍了目前有潜力用于治疗中枢神经系统疾病的核酸药物及其作用机制; 概述了BBB作为核酸药物递送的主要障碍, 穿越BBB的主要方法及相关机制机理; 重点讨论了各种递送系统(如病毒载体、核酸药物偶联、脂质纳米颗粒、聚合物纳米颗粒和外泌体等) 的特性及它们在介导核酸药物脑部递送方面的相关进展。

1　核酸药物的分类及其作用机制

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核酸药物的作用机制根据其生物学效应可分为3个主要类别: 下调基因表达、上调基因表达和基因编辑。小干扰核糖核酸(small interfering RNA, siRNA)、小分子核糖核酸/微核糖核酸(microRNA, miRNA) 和反义寡核苷酸(antisense oligonucleotides, ASOs) 是通过与靶RNA配对来抑制靶基因的典型核酸药物。mRNA和质粒脱氧核糖核酸(plasmid DNA, pDNA) 通常用于增加靶基因的表达。而成簇的规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein, CRISPR/Cas) 系统可以增加、抑制或纠正靶基因的表达。

1.1　siRNA

siRNA也叫作沉默RNA, 通常以双链形式存在, 单链形式发挥作用, 由21到25个核苷酸组成, 3′端一般有两个游离的碱基。它可以由长的双链RNA或小发夹RNA (small hairpin RNA, shRNA) 经Dicer酶(一种特殊核糖核酸酶, 属于RNase Ⅲ家族) 特异性识别并切割形成(内源性)^[17], 也可以由化学合成的方法转染到细胞中(外源性)。siRNA可以通过RNA干扰(RNA interference, RNAi) 机制, 特异性地促进靶细胞质中靶mRNA降解或抑制其翻译表达, 产生基因沉默效应^[2]。

在细胞质中, siRNA与RNA诱导沉默复合物(RNA-induced silencing complex, RISC) 结合后, 解旋成单链。正义链被降解, 而与目标转录本(完全) 互补的反义链作为导向子(guide strand) 留在RISC上, 形成活性的RISC-siRNA复合物。一旦RISC-siRNA复合物与目标mRNA相互匹配, RISC上具有核酸内切酶活性的Argonaute蛋白将引导该mRNA的降解或阻止其翻译成蛋白质(图1A)。通过这一机制, siRNA能够精准沉默目标基因的表达, 在细胞内发挥调控作用。这种基因沉默效果是可逆的, 只要停止给予siRNA药物, 靶基因就会恢复表达。siRNA可以针对任何编码蛋白质或非编码RNA的基因, 具有高效率和高度特异性, 目前已有多种siRNA药物获批上市。其中, ONPATTRO是FDA批准的首款siRNA药物, 用于治疗遗传性转甲状腺素蛋白淀粉样变性伴多发性神经病, 是RNAi领域的一个里程碑事件。

1.2　miRNA

miRNA是一类内源性非编码单链RNA分子, 长度约为19~24个核苷酸。miRNA在细胞内通过参与RNAi机制来调控基因表达, 从而影响细胞的生理过程。miRNA的作用机制与siRNA类似, 但也有一些独有的特征。miRNA可通过与靶mRNA的3′非编码区(untranslatd region, UTR) 或编码区互补结合, 导致靶mRNA的降解或翻译抑制, 从而影响细胞的生长、分化、凋亡、代谢等多种生理过程^[18]。内源性的pre-miRNA在进入到细胞质中后由Dicer切割成约22个核苷酸的不完全配对的双链RNA, 一条链(passenger strand) 降解, 另一条链作为guide strand与Argonaute蛋白结合形成RISC^[19]。RISC通过miRNA的5′端与靶基因mRNA的3′UTR部分互补结合, 抑制目标mRNA的翻译或促进其降解(图1A)。这一过程导致目标基因的表达下调, 实现了miRNA在基因调控中的功能^{[20, 21]}。siRNA与miRNA都是通过RNAi的方式来发挥作用, 但是也存在一些不同。miRNA来源于较短的茎环RNA产物, 主要作用于靶标基因的3′UTR区, 可以通过不完全配对抑制翻译或者降解mRNA来沉默基因, 在翻译水平和转录后水平发挥作用, 具有更广泛的调控范围。而siRNA具有100%的互补性, 通常进行转录后水平的调控, 即通过在翻译前切割mRNA导致靶标基因的降解, 具有更高的特异性。

1.3　ASOs

ASOs是人工合成的单链核酸聚合物, 通常由18~30个核苷酸组成。它们与目标基因的特定RNA序列互补结合, 通过干扰mRNA功能实现治疗效果(图1B)。ASOs主要通过3种机制调控基因表达: 降解mRNA (招募RNase H切割降解mRNA, 阻断蛋白质合成); 抑制翻译(ASOs与mRNA结合, 阻止mRNA与核糖体结合, 降低蛋白质合成); 调控剪接(ASOs结合于pre-mRNA的外显子区域, 使外显子被剪切掉, 纠正基因剪接异常)。ASOs因其高度特异性在治疗基因相关疾病, 如脊髓性肌萎缩症(spinal muscular atrophy, SMA) 和遗传性视网膜病变等方表现出色^{[22, 23]}。

1.4　mRNA

mRNA是一种单链长核糖核酸, 传递DNA中的遗传信息并翻译合成蛋白质(图1C)。其结构包括5′端帽子结构, 保护mRNA免受降解并促进转运和翻译; 5′端UTR, 调节mRNA稳定性和翻译效率; 编码序列, 翻译成蛋白质; 3′端UTR, 影响mRNA稳定性和降解; 以及多聚腺苷酸尾巴, 保护mRNA并增加其转运和翻译效率。

mRNA在核酸药物疗法、诊断生物标志物和治疗靶标等方面应用广泛。细胞内的mRNA可以翻译产生治疗性蛋白, 替代有缺陷或缺失的蛋白。进入体内的mRNA由细胞表达特定蛋白, 避免体外因素影响, 并调节免疫系统, 消除包括癌细胞在内的威胁。编码抗原序列的mRNA疫苗通过脂质纳米载体递送进入细胞, 由人体细胞翻译产生抗原, 激活免疫反应。mRNA疗法主要应用于传染病疫苗、治疗性癌症疫苗、免疫肿瘤学疫苗和蛋白质替代等。

1.5　pDNA

pDNA是一种环状双链DNA分子, 可以独立于染色体或核区复制, 主要存在于细菌、放线菌和真菌细胞中。它通常包含一个或多个外源基因及其调控元件, 如启动子、终止子和选择性标记基因。通过转染等方法, pDNA可将外源基因导入目标细胞, 随后被转录成mRNA并翻译成蛋白质, 用于纠正或补充缺失或异常基因(图1C)。作为基因载体, pDNA具有自主复制和转录的能力, 在子代细胞中保持恒定拷贝数, 表达所携带的遗传信息。pDNA因其安全、低毒、低免疫原性和易于分离提取等优点, 广泛应用于基因治疗、细胞治疗和核酸疫苗等领域。

基于pDNA的基因治疗是将治疗基因整合到质粒载体中, 通过各种递送方式(如电穿孔、脂质体、病毒载体等) 将质粒导入靶细胞, 从而实现基因表达以治疗疾病的一种基因治疗策略^[24]。DNA疫苗是将编码特定抗原的基因序列插入质粒载体中, 通过注射使机体细胞表达目标抗原, 从而诱导机体产生免疫应答的一种新型疫苗技术^[25]。相较于mRNA, pDNA更具成本效益、可运输性和稳定性^[26]。然而, 其缺点也十分明显, 主要是转染效率低且表达起效慢, 因为pDNA需要跨越核膜进入细胞核才能发挥作用, 且需要经过转录和翻译两个步骤才能产生蛋白质。此外, pDNA还存在潜在的基因组整合风险和免疫原性问题, 其较大的分子质量也增加了细胞递送的难度^[27]。

1.6　CRISPR/Cas

CRISPR/Cas是一种存在于原核生物(细菌和古细菌) 的免疫系统, 能够在遭到病毒入侵后提取出病毒的一小段DNA存储到自身基因组的特定区域上(CRISPR存储空间)。当再次遇到病毒入侵时, 原核生物根据存储的DNA识别并切割病毒DNA。

CRISPR/Cas系统包含CRISPR基因座和Cas基因两部分。CRISPR基因序列包括前导序列(leader)、重复序列(repeat) 和间隔序列(spacer) (图1D)。前导序列位于CRISPR基因上游, 引导重复和间隔序列的转录; 重复序列含20~50个碱基对, 形成稳定RNA的发卡结构; 间隔序列存储细菌从外源DNA捕获的片段, 提供免疫记忆。Cas基因编码与CRISPR序列共同作用的蛋白, 包括Cas1到Cas10, CRISPR/Cas系统分为两类: 多蛋白复合物的第一类(Ⅰ、Ⅲ、Ⅳ型) 和依赖单一Cas蛋白的第二类(如Ⅱ型Cas9和V型Cpf1)^[28]。最常用的是CRISPR/Cas9系统, 由sgRNA和Cas9蛋白组成, sgRNA识别PAM序列, 激活Cas9切割DNA, 导致双链断裂, 通过NHEJ或HDR修复^[29]。Cas9蛋白和sgRNA可以共编码到同一pDNA中、作为mRNA或核糖核蛋白复合物(ribonucleoprotein complex, RNP) 使用。例如, Park等^[30]利用RNP结合两亲性R7L10肽, 靶向神经元中的BACE1基因, 降低β淀粉样斑块(β-amyloid plaques, Aβ) 42分泌, 用于治疗AD。尽管CRISPR/Cas9技术在疾病模型开发和遗传病治疗方面具有良好的应用前景, 但可能导致正常基因功能的破坏或导致基因组不稳定的脱靶效应仍然是一个严重的问题^[31]。

1.7　适配体

适配体是完全人工设计和合成的单链DNA或RNA寡核苷酸, 具有特定的三维结构, 通过氢键、静电相互作用和范德华力与目标分子相互作用。大多数适配体通过指数富集配体系统筛选得到。与其他以核酸为基础的疗法不同, 适配体不仅通过互补碱基配对识别特定靶标序列, 还可以形成三维二级结构与蛋白质相互作用来实现靶向。通过阻断关键分子、诱导降解、递送药物和调节信号传导等多种方式, 它们能够有效调控生物过程, 干预疾病进程(图1E)。适配体药物在抗癌、抗病毒、抗炎和免疫调节等领域展示了广泛的应用潜力, 且具有高特异性、低免疫原性和易于修饰等优势。Cheng等^[32]开发了靶向适配体并探索其杀死胶质瘤细胞的机制。研究表明, AS1411与胶质瘤细胞中过表达的核仁素结合, 诱导p53上调和Bcl-2下调, 随后引发细胞凋亡和周期停滞, 展示了其在胶质瘤治疗中的潜力。由于其特殊结构, 适配体还可以用作靶向配体。例如, AS1411与透明质酸双功能化的微乳剂可穿透BBB, 靶向胶质瘤并递送shikonin和docetaxel^[33]。这种制剂能够选择性地在U87胶质瘤细胞中积累, 抑制肿瘤生长和癌症干细胞的形成。然而, 体内稳定性和递送效率仍是其进一步发展的挑战。

与传统的小分子和蛋白质药物相比, 核酸药物在治疗靶点范围和靶点特异性方面表现出更显著的优势, 且其治疗效果通常更为持久。截至目前, 在核酸药物开发领域, 已有9款ASOs、6款siRNA、2款mRNA疫苗和2款RNA适配体药物获得批准上市。其中nusinersen可以纠正SMN基因的剪接缺陷, 增加SMN蛋白的产生, 进而改善SMA患者的神经系统功能和运动能力, 而tofersen则用于治疗由SOD1基因突变引起的肌萎缩侧索硬化(amyotrophic lateral sclerosis, ALS)。如表1所示, 还有多款针对中枢神经系统疾病的核酸药物正在临床试验阶段, 进一步证明了核酸药物在这个治疗领域中的巨大潜力。

2　核酸药物脑部递送遇到的障碍

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基于RNA或DNA的基因治疗及基于CRISPR的治疗性基因组编辑虽然为中枢神经系统疾病的治疗提供了潜在的希望, 但核酸类药物递送至中枢神经系统的过程中面临着多重生理屏障的挑战。这些障碍包括循环系统中的降解和清除、BBB、细胞摄取效率、内体/溶酶体屏障及药物释放等问题, 这些因素共同制约了核酸药物向大脑的有效递送(图2A~E)。

2.1　循环中的降解与清除

经静脉给药后, 核酸药物随血液循环分布至全身(图2A), 在此过程中, 核酸药物在体内的清除途径和生物分布表现出明显的尺寸依赖性。具体而言, 小于6 nm的核酸药物制剂主要通过肾小球滤过单元被迅速清除; 而中等尺寸的制剂(20~150 nm) 则倾向于在肝脏中被单核巨噬细胞(mononuclear phagocyte, MPS) 捕获并清除。MPS由一系列细胞组成, 包括肝窦内皮细胞及库普弗细胞, 其中库普弗细胞作为常驻的巨噬细胞, 具有强大的吞噬能力, 它们与内皮细胞共同作用, 能够捕获并清除肝脏中超过90%的核酸药物^[34]。而超过200 nm的核酸制剂对MPS的清除具有较高的抵抗力, 并会在肝脏、脾脏和肺部积累。

血液循环中的核酸药物制剂与血浆蛋白之间的非特异性结合对其体内分布和治疗效果构成了重大挑战。核酸因其固有的负电荷, 易于与血浆蛋白(例如白蛋白和球蛋白) 非特异性结合。这种非特异性结合可能会降低药物的有效浓度, 并加速药物在体内的清除过程^{[35, 36]}。此外, 将核酸药物封装在纳米载体中进行递送时, 这些载体在血液循环中也会吸附血浆蛋白形成蛋白冠。蛋白冠的形成会改变纳米载体的表面特性, 包括化学组成、电荷和亲疏水性, 进而影响其在体内的分布、细胞摄取及免疫反应^[37]。而这尤其影响核酸药物脑部递送常采用“主动靶向”策略的纳米载体。这类载体表面常设计有小分子、肽段和抗体片段等配体以提高其脑靶向能力, 而其表面特性的改变可能会通过电荷特异性或分子间的相互作用进一步促进蛋白冠的形成, 由于冠层掩盖了靶向配体, 纳米载体的组织归巢效率会大幅下降^{[38, 39]}。例如, Salvati等^[38]发现, 转铁蛋白修饰的聚乙二醇化二氧化硅纳米粒子经胎牛血清处理后在表达转铁蛋白受体的肿瘤细胞中的摄取减少, 靶向性降低。但在无血清培养基或磷酸盐缓冲液中, 它们能高效地通过转铁蛋白受体介导的内吞作用被细胞摄取。

由此可见, 核酸药物制剂在体内应尽量避免核酸酶降解、肝脏和肾脏快速清除及与血浆蛋白的非特异性结合, 才能维持其稳定性和疗效^[40]。此外, 进一步优化纳米载体的设计, 以减少蛋白冠的形成并保持其靶向配体的活性, 对于提高核酸药物在脑部的递送效率至关重要。

2.2　血脑屏障

BBB是一种具有高度选择性的半透膜结构, 通过将血液与大脑细胞外液隔离来维持脑内环境稳定, 对大脑起着至关重要的保护作用。其结构主要由脑血管内皮细胞(endothelial cells, ECs)、基底膜(basement membrane)、周细胞(pericyte) 和星形胶质细胞(astrocyte) 的终足(end foot) 组成(图2G)。作为BBB的核心结构, ECs构成脑血管内层, 通过细胞间紧密连接限制水溶性及大分子物质通过, 并表达特殊转运蛋白和外排转运蛋白来调控物质转运。周细胞环绕毛细血管内皮细胞, 嵌入基底膜, 参与调控脑血流量与BBB通透性^[41]。星形胶质细胞的末梢环绕脑血管, 经终足连基底膜, 其确切作用虽存争议, 但目前公认它能与内皮细胞、周细胞共同维持BBB功能^[42]。基底膜位于上述细胞之间, 为邻近的细胞提供结构与功能支持, 还能通过与细胞表面受体作用调节细胞行为与信号传导^[43]。上述组成部分协同构成高选择性屏障, 既保障必需物质进入, 又阻挡了有害物质, 但也给核酸药物入脑带来挑战。BBB阻碍了98%小分子(< 500 Da) 及几乎所有大分子(> 1 kDa) 神经治疗药物的穿透, 极大地增加了中枢神经系统疾病的治疗难度^[44], 因此开发能够有效突破BBB的递送策略成为当前研究的重要方向^[45]。

2.3　进入靶细胞

当核酸药物达到目标细胞的表面, 首先需要进入细胞(图2C)。核酸药物进入靶细胞的主要途径有吞噬作用、巨胞饮作用、网格蛋白介导的内吞作用、小窝依赖性内吞作用和网格蛋白/小窝非依赖性内吞作用等^[46-48]。由于其分子质量大且带负电, 核酸药物难以直接穿过细胞膜进入细胞, 为了提高细胞摄取效率和逃避内体/溶酶体的降解, 研究人员开发了多种递送方法。一方面通过化学修饰, 如2′-O-甲基化、硫代磷酸酯修饰等, 可以提高核酸药物的稳定性和细胞膜渗透性; 另一方面, 利用纳米载体可以实现核酸药物的精确靶向和目标细胞对其的高效摄取。通过将核酸药物包裹在纳米载体颗粒中, 以提高其稳定性和细胞摄取能力, 同时通过表面修饰配体(主要是小分子与肽) 与细胞膜上的特异性受体结合精准靶向病变细胞, 用更少的剂量获得有效的治疗效果并减少不良反应。例如, 叶酸是一种典型的小分子配体, 它能够与肿瘤细胞膜上高度表达的叶酸受体特异性结合, 常用于治疗脑肿瘤^{[49, 50]}。甘露糖修饰的载体可以与小胶质细胞上的甘露糖受体结合^[48], Tet1肽(HLNILSTLWKYR) 对神经元上的三唾液酸神经节苷脂具有高亲和力, 这使得它们成为治疗神经退行性疾病的有力工具^{[51, 52]}。

2.4　内体/溶酶体屏障

核酸药物及其纳米载体经细胞摄取后通常会经过内体/溶酶体途径(图2D), 由于溶酶体内的酸性环境(pH 4~5) 和降解酶, 核酸药物易发生降解, 转染效率降低^[53]。目前主要有3种方法克服内体/溶酶体屏障, 包括被动地破裂内体/溶酶体、主动诱导内体/溶酶体结构变化, 以及在内吞作用过程中绕过内体/溶酶体途径。

利用质子海绵效应来促使内质网/溶酶体破裂是核酸药物递送中的一种常见的逃逸策略。含有伯胺、仲胺和叔胺等碱性基团的聚合物能够吸附氢质子, 从而显著增加内质网/溶酶体内的渗透压, 导致内质网/溶酶体吸水膨胀并破裂, 释放出药物制剂。富含伯胺、仲胺和叔胺基团的聚乙烯亚胺(polyethyleneimine, PEI) 是最典型的具有质子海绵效应的聚合物, 被广泛用于递送核酸药物, 然而, 使用PEI时也需注意其潜在的毒性问题^[54]。除了PEI, 其他多种阳离子聚合物也被用于促进核酸药物的传递并帮助其从内体/溶酶体中逃逸, 例如聚赖氨酸(含伯胺)、聚(L-组氨酸) (含咪唑基团)、聚精氨酸(含伯胺和仲胺) 及聚(β-氨基酯) (含叔胺)。狂犬病病毒糖蛋白肽(rabies virus glycoprotein peptide, RVG) 29-9R肽是一种通过在RVG29分子的羧基末端添加含有9个精氨酸残基构建而成的, 它能够携带核酸药物, 并通过质子海绵效应促进内体/溶酶体的逃逸, 同时保持RVG29的原有功能不受影响^{[55, 56]}。这些研究进展为核酸药物的递送提供了新的思路和方法。

目前也有多种载体的逃逸机制是通过与内体/溶酶体的相互作用诱导其结构破坏。Shi等^[57]通过将疏水性光敏剂TBD与聚乙二醇(polyethylene glycol, PEG) 修饰, 合成了一种两亲性聚合物TBD-PEG-N3。该聚合物能够自组装为聚合物纳米颗粒, 并通过点击化学与二苯并环辛炔修饰的ASO结合。在光照条件下, 光敏剂TBD产生大量单线态氧, 破坏内体/溶酶体的结构, 促进药物的释放。在常用的脂质纳米颗粒核酸递送系统中, 阳离子脂质被认为与内体逃逸密切相关。阳离子脂质一般是由一个极性头部通过连接子和疏水的尾巴相连接。目前新一代的阳离子脂质包含了可降解的可离子化脂质, 其pK_a值一般在6.0~7.0之间, 在生理条件下呈现电中性。当进入内体的酸性环境时, 可离子化脂质的头部会被质子化, 带上正电荷, 与内体膜上的阴离子脂质相互作用, 这一过程促使脂质体形成反六角相结构, 通过膜融合或膜破裂释放有效载荷进入细胞质中^[58]。

近年来, 能够绕过内体/溶酶体途径的细胞摄取方式引起了广泛关注, 其中外泌体包裹可以通过膜融合介导载体的摄取, 从而绕过内体/溶酶体途径。研究发现, 直径40~100 nm的突触小泡(一种脂质双层结构) 能通过与质膜融合来释放神经递质和激素^{[59, 60]}, 这一过程主要由可溶性N-乙基马来酰胺敏感因子附着蛋白受体(soluble N-ethylmaleimide-sensitive factor attachment protein receptors, SNAREs) 蛋白介导; 通过将SNAREs来源的跨膜片段syb (TMS-syb) 插入人工脂质双层, 成功模拟了突触小泡的膜融合功能, 促进载体通过膜融合进入神经元, 绕过了内体/溶酶体途径^[61]。You团队^[62-65]发现由33个氨基酸组成的多肽pardaxin能够介导脂质体通过小窝介导的内吞作用进入细胞并靶向内质网, 不仅避免了核酸药物在内质体/溶酶体中被核酸酶降解, 还有利于pDNA或CRISPR/Cas9系统进入细胞核进行基因治疗。

2.5　药物释放

核酸药物通常在细胞质或细胞核中发挥作用, 其从载体上有效释放是其发挥作用的前提。内体/溶酶体中的酸性微环境可以作为内源性刺激来促进核酸药物释放。Zhou等^[66]合成了(DPAx-co-DMAEMAy)-PG三嵌段pH敏感多聚阳离子, 将其组装成胶束并通过静电吸附siRNA。在酸性条件下, DPA由于质子化从疏水性转变为亲水性, 促使胶束降解并释放siRNA。细胞内的各种酶也是药物释放的理想内源性刺激。例如酯酶就是一种广泛存在于细胞质中的水解酶, 可以通过催化酯键的水解来介导化学药物的释放。在脑肿瘤细胞质中特异性高表达的谷胱甘肽(glutathione, GSH) 可以通过还原效应促进核酸药物的释放。Wu等^[67]合成了对GSH敏感的嵌段共聚物, 通过GSH介导的二硫键断裂, 促进了药物的释放。肿瘤和神经退行性疾病中活性氧(reactive oxygen species, ROS) 水平升高, 可以利用这一特点来刺激药物的释放。例如Liu等^[68]合成了带有苯硼酸的正负电荷转换聚合物, ROS可以降解苯硼酸, 从而将聚合物转化为聚丙烯酸, 通过静电排斥释放DNA。此外, 通过膜融合直接将核酸药物释放到细胞质中也是一种高效且简便的递送策略。载体与细胞膜的直接相互作用是其核心机制, 通常通过电荷作用(载体通常带有阳离子, 可以与带负电的细胞膜相互吸引) 或疏水性相互作用来触发载体膜与细胞膜之间的相互作用, 促使磷脂双层的重排和融合, 从而在无需进入内体/溶酶体的情况下释放核酸药物至细胞质。这种方法可以避免核酸药物在内体/溶酶体内被核酸酶降解的风险, 提高了其在靶细胞内的稳定性和有效性。

3　核酸药物脑部递送的途径及其机制

收起

3.1　绕过BBB的方式

3.1.1　鞘内注射和脑室内注射给药

药物中枢递送的最直接方法是将药物注射到脑脊液中。这种方式可绕过BBB, 提高药物在大脑中的浓度并减少全身不良反应, 主要的给药方式有鞘内注射(intrathecal, IT) 和脑室内注射(intracerebroventricular, ICV)。IT给药通过腰椎穿刺或植入鞘内药物递送装置进行, 将药物注入脊髓周围的鞘内或蛛网膜下腔^{[69, 70]}, 这种方法有助于药物分布到脊髓、小脑和大脑表面, 是治疗神经和神经肌肉疾病的ASO/siRNA最常用的给药途径^[71]。ICV给药则是通过穿过颅骨将药物注射到侧脑室, 药物通过脑脊液循环分布于整个大脑。两种给药方式广泛应用于需要高浓度药物在脑内的中枢神经系统疾病治疗, 如脑肿瘤、神经退行性疾病和某些中枢神经系统感染等。

Nusinersen和tofersen是两种已获FDA批准的小核酸药物, 均采用IT给药的方式。它们属于ASOs, 具有单链结构, 并在磷酸骨架中富含磷酸硫代(phosphorothioate, PS) 修饰。PS修饰提高了寡核苷酸的稳定性和亲脂性, 使其具有跨越细胞膜的能力, 增强了其在体内的分布和生物利用度。siRNA相比ASOs更为亲水, 并带有较多负电荷, 直接跨膜能力较弱, 需要通过化学手段提高其疏水性以用于局部注射。Alnylam公司的ALN-APP (alnylam amyloid precursor protein) 是这一技术的代表分子。近期, 该公司公布了通过IT给药ALN-APP治疗AD和脑淀粉样血管病(cerebral amyloid angiopathy, CAA) 的Ι期临床试验的中期结果。ALN-APP采用C16脂肪酸链偶联的方式, 增加了siRNA的疏水性, 辅助其跨越细胞膜, 从而发挥沉默靶基因的作用。APP基因的突变是导致早发性阿尔茨海默病和脑淀粉样血管病的重要原因, 且淀粉样蛋白沉积是阿尔茨海默病患者大脑的标志性特征之一。通过靶向编码APP蛋白的mRNA, ALN-APP旨在降解mRNA, 减少APP蛋白的表达, 从而治疗AD和CAA。初步结果显示, 单剂量ALN-APP能在患者体内快速结合靶标, 并持续降低sAPPα和sAPPβ水平超过6个月。

3.1.2　鼻内给药

与IT和ICV相比, 鼻内给药(intranasally, IN) 是一种非侵入性途径, 主要通过嗅觉神经和三叉神经通路, 避开BBB将核酸药物直接递送到大脑^[72]。其中, 嗅觉神经是最重要的途径, 利用其传输功能可有效绕过BBB^[73]。嗅觉通路主要以神经元外(神经细胞外间隙) 转运和神经元内(轴突内) 转运两种方式将药物递送到CNS^[74]。前者是通过药物在嗅觉神经束的细胞外间隙的扩散来实现的, 药物分子可以在鼻黏膜上皮通过细胞外途径进入, 然后通过嗅球进入大脑, 这种转运方式的速度较快, 可以在几分钟到半小时内完成。后者则涉及药物通过被动扩散、吸附性内吞和受体介导的内吞等细胞内机制进入细胞, 通过嗅觉神经元的轴突进行转运。药物分子在与嗅觉上皮的嗅觉感受神经元结合后, 可以沿着神经元的轴突进入大脑, 最终到达嗅球和其他脑区, 这种转运方式的速度较慢, 可能需要数小时到数天^[73]。一旦药物到达嗅球和脑脊液, 它们可以通过与脑细胞外液的混合进入CNS的其他区域。

三叉神经通路是实现鼻-脑药物递送的另一种途径。药物可以通过这一通路穿过呼吸和嗅觉上皮, 通过细胞内或细胞外的运输机制沿三叉神经进入大脑^[75]。这种方式可以快速实现药物从鼻腔到大脑的靶向治疗^{[76, 77]}。此外, 药物还可以通过血液循环通路进入CNS。具体来说, 药物可以通过鼻腔黏液层、鼻上皮细胞层进入静脉血流, 随后穿透BBB进入CNS^{[78, 79]}。

经鼻给药可快速直接进入CNS, 减少药物在血液循环中的延迟和清除, 降低非靶向器官(如肝脏、脾脏和肾脏) 的毒性风险^[80]。Rohn等^[81]通过非侵入性鼻腔递送腺相关病毒9型(adeno-associated virus serotype 9, AAV9) 载体, 绕过BBB, 将CRISPR/Cas9基因编辑系统导入小鼠CNS, 成功下调了HTR2A基因表达, 显著减少5HT-2A受体的表达, 改善了小鼠的焦虑行为。

目前, 鼻内给药也存在一些挑战: 需要最大化CNS渗透并最小化肺部暴露; 鼻纤毛的清除和酶活性会限制药物在鼻腔中的停留时间和稳定性; 未修饰的核酸药物易被鼻腔中的核酸酶降解或引发免疫反应; 鼻腔递送只能进行低剂量给药, 需要频繁滴入; 存在剂量变化和个体吸收特性的差异等^[82]。为了克服这些挑战, 研究者们正在探索纳米载体的表面修饰, 以提高药物的靶向递送效率和内体逃逸能力。相关研究表明, 提高内体逃逸效率可以增加核苷酸药物在细胞质中的浓度^[83]。尽管存在一些限制, 鼻腔给药途径凭借其直接的鼻脑转运通路、丰富的血管分布、多孔的内皮基底膜及能够绕过肝脏首过效应等优势, 显示出较高的临床应用潜力^[84]。

3.1.3　对流增强递送

对流增强递送(convection-enhanced delivery, CED) 是一种用于将药物直接递送到脑组织中的局部给药技术。该方法通过在压力梯度下持续注射药物, 使药物通过细胞间的空隙进入大脑组织, 从而绕过BBB, 实现高效的局部药物递送。Cohen等^[85]采用CED技术, 通过颅内注射(intracranial injection, IC) 将透明质酸修饰的脂质纳米颗粒直接递送到脑部肿瘤位置。这些纳米颗粒包裹着靶向PLK1基因的siRNA, 能够选择性结合肿瘤细胞上高表达的CD44受体, 从而实现特异性靶向作用。通过CED, 药物可以绕过BBB, 在肿瘤部位形成高浓度药物分布, 有效抑制PLK1表达, 促进肿瘤细胞凋亡和抑制肿瘤生长。

3.2　克服血脑屏障

3.2.1　物理方法

聚焦超声(focused ultrasound, FUS) 结合微气泡(microbubble, MB) 技术是一种非侵入性的物理手段。该技术通过将低频超声能量聚焦于特定脑区, 与预先注入血液循环中的MB协同作用, 短暂且局部地打开BBB^[86]。在超声波的作用下, MB发生振荡, 产生机械力作用于内皮细胞的紧密连接, 在约1~4 h内创建一个药物递送窗口, 窗口在约24 h后恢复^[87]。该过程能够在不破坏神经组织的前提下, 促进核酸药物的局部渗透。该方法的优势在于其空间分辨率高, 可精确定位于目标脑区, 且BBB的打开是可逆的。Guo等^[88]探讨了微气泡增强的聚焦超声(FUS-MB) 结合50 nm大小的阳离子脂质-聚合物混合纳米颗粒能高效递送siRNA到脑肿瘤微环境中, 显著抑制Smoothened蛋白的表达, 并诱导肿瘤细胞凋亡。Kwak等^[89]通过FUS-MB暂时性地打开BBB, 使系统性注射的纳米颗粒穿过BBB并精准递送至目标脑区。研究中使用的聚(β-氨基酯) 纳米颗粒经过表面修饰, 能够稳定包裹核酸药物(如pDNA和mRNA), 并在神经胶质细胞和神经元中实现报告蛋白表达和CRISPR/Cas9基因编辑。同时磁共振成像(magnetic resonance imaging, MRI) 可以提供精确定位, MRI引导的FUS-MB能够精确打开肿瘤^[90]和纹状体^[91]区域的BBB, 分别应用于治疗胶质瘤和帕金森病。然而, FUS-MB可能因增加局部温度及机械效应而损伤血管, 并允许有毒物质进入中枢神经系统, 因此需优化FUS参数以减少脑损伤^[92]。超短脉冲激光也能通过类似原理局部打开BBB且不破坏血管完整性^[93]。全脑高温疗法虽能提高BBB通透性, 但可能会对大脑造成严重的伤害, 包括细胞损伤和功能障碍, 具有潜在的严重不良反应和风险^[94]。

电穿孔技术通过在BBB上施加短暂的高强度电场, 诱导血管内皮细胞膜极化, 形成微小孔洞, 瞬时提高BBB的通透性, 使得治疗性核酸能够进入脑组织, 促进核酸药物的脑部递送^{[95, 96]}。更强、更长的电脉冲可以形成更多的孔, 从而得到更高的转染速率, 但是这也可能导致外部介质扩散到细胞中, 增加毒性, 进而引起细胞死亡、组织损伤和炎症反应等不良反应。因此, 在应用电穿孔技术时, 需要精确控制电场强度和脉冲时间等参数, 以平衡转染效率和细胞损伤。

3.2.2　化学方法

此外, 一些生物和化学物质(如封闭带毒素、组胺、缓激肽、油酸等) 也可暂时打开BBB^[97]。Lexiscan是一种能够短暂增强BBB通透性的化学物质, 它通过结合并激活血管内皮细胞上的腺苷A2A受体, 引起血管扩张并增加局部血流量, 暂时性地松解BBB的紧密连接。这种机制使得BBB的通透性短暂增强, 使大分子药物或纳米颗粒能够更容易穿越屏障, 进入脑组织进行治疗。Lexiscan的作用是短暂且可逆的, 能够增加药物在脑部靶向区域的渗透和累积, 尤其在脑肿瘤(如胶质母细胞瘤) 的治疗中, 显著提高了递送效率^[98]。

3.3　跨越BBB

亲脂性药物和一些特殊设计的药物可通过跨细胞转运途径穿过BBB。这包括通过血管内皮细胞的内吞作用、囊泡运输和外排作用等。跨细胞途径允许药物直接穿过细胞膜, 是一种更为有效的药物递送方式, 具有更大的潜力。它可分为3个不同的类别: 受体介导的转运(receptor-mediated transcytosis, RMT)、载体蛋白介导的转运(carrier-mediated transcytosis, CMT) 和吸附介导的转运(adsorptive-mediated transcytosis, AMT)。RMT通过特定受体介导的通路进行核酸药物的转运。跨越BBB的RMT过程需要配体与其在脑微血管和毛细血管内皮细胞腔膜上的同源受体结合, 并确保靶受体蛋白在脑血管内皮细胞中高表达, 而在外周血管中最低表达, 从而实现药物的有效递送^[92]。CMT通过特定的膜载体蛋白介导核酸药物跨BBB转运^[99]; 而AMT则通过富含精氨酸或赖氨酸等阳离子氨基酸的细胞穿透肽(cell-penetrating peptides, CPPs) 经静电吸附内皮细胞来增强BBB通透性^[100]。

3.3.1　受体介导的胞吞转运

在受体介导的转胞吞作用中, 特定的配体与内皮细胞管腔侧的特异性受体结合, 触发内吞作用形成转胞囊泡, 转运到大脑。这种方法是研究最广泛的脑递送途径^[101]。常见的受体包括转铁蛋白受体(targeting transferrin receptor, TFR)、烟碱乙酰胆碱受体(nicotinic acetylcholine receptor, nAChR)、胰岛素受体和低密度脂蛋白受体相关蛋白(low-density lipoprotein receptor-related protein, LRP) 等。

3.3.1.1　TFR

TFR在脑毛细血管内皮细胞表面高度表达, 通过受体介导的内吞作用内化转铁蛋白结合的铁。大多数用于RMT的配体是序列特异性肽, 它们因合成成本低且易于大规模生产而受到青睐。同时这些肽还具有氨基、羧基和巯基等反应性基团, 可以在温和反应条件下将其化学修饰到纳米粒子表面。目前已开发出一种标准的载体修饰肽方法, 即通过Michael加成反应, 将特定序列肽的半胱氨酸修饰后共价附着到马来酰亚胺修饰的纳米粒子上。特定序列肽如T7 (HAIYPRH)、TBP (GGGHKYLRW) 等已被开发出来, 它们可以通过特异性结合BBB上的转铁蛋白受体来增加核酸药物在大脑中的积累^{[102, 103]}。

靶向BBB上的转铁蛋白受体还可以通过在纳米颗粒表面修饰80 kDa的转铁蛋白来实现。许多研究表明, 结合有转铁蛋白的纳米系统能够通过蛋白-受体结合和随后转铁蛋白受体介导的转胞吞作用有效地穿越BBB, 将药物递送到大脑^{[104, 105]}。Rodrigues等^[106]开发了转铁蛋白修饰的纳米系统, 用于将核酸治疗物递送至大脑。他们首次开发了一种CPP渗透素(penetratin, Pen) 和转铁蛋白结合的脂质体双配体功能化载体, 以跨BBB递送pDNA^[106], 在体外BBB模型(由脑内皮细胞bEnd.3和大鼠原代星形胶质细胞构建) 中, 双配体脂质体的效率显著高于单配体脂质体(7%对比3%GFP阳性神经元)。体内研究显示, 相比于未结合或单一配体功能化脂质体。静脉注射的双配体脂质体在小鼠大脑中转染效率更高^[106]。Rodrigues等^[107]后来扩展了他们早期的工作, 在小鼠AD模型中测试了这些脑靶向CPP-转铁蛋白脂质体的治疗潜力和有效性, 研究发现, 静脉注射负载神经生长因子基因pDNA的CPP-转铁蛋白脂质体显著减少了AD小鼠脑中的Aβ沉积。

3.3.1.2　nAChR

nAChR在中枢神经系统中高度表达且分布广泛, 可以利用其相关的配体通过RMT实现脑靶向。例如来自狂犬病毒糖蛋白的RVG肽含有29个氨基酸, 可以结合脑毛细血管内皮细胞和神经元上的nAChR。RVG肽可以诱导受体介导的转胞吞作用, 穿透BBB, 最终将生物制剂运输到脑实质, 目前已成功作为靶向大脑的配体, 用于递送小分子药物、siRNA和蛋白质进行诊断和/或治疗, 适用于各种疾病, 如多形性胶质母细胞瘤(glioblastoma multiforme, GBM)、创伤性脑损伤和AD等^{[108, 109]}。

Shen等^[110]报道RVG肽能够促进CRISPR/Cas9递送至大脑, 他们介绍了一种通过一步合成法构建的可追踪纳米生物杂化复合物(fluorescent traceable biohybrid iron oxide, F-TBIO), 用于AD的CRISPR/Cas9基因编辑与化学药物协同治疗。F-TBIO由超顺磁性氧化铁纳米颗粒(superparamagnetic iron oxide nanoparticle, SPION) 核心构建, 并与聚赖氨酸-PEG连接, 末端为RVG或氟伐他汀。这些纳米系统能够穿越BBB并靶向神经元, 有效递送CRISPR/Cas9基因编辑器和药物。体内研究表明, 该系统可以显著减少AD小鼠海马体中的Aβ斑块面积, 并改善其认知能力。同时, F-TBIO具有良好的MRI成像性能, 能在复杂的脑生理环境中提供准确的成像信号, 展示了其作为神经退行性疾病治疗平台的巨大潜力。

3.3.1.3　LRP

LRP (如LRP1和LRP2) 在中枢神经系统(包括脑内皮细胞和GBM细胞) 中高度表达^[111], 介导的多种配体[包括载脂蛋白E (apolipoprotein E, ApoE) 和某些神经保护因子], 被广泛用于研究神经退行性疾病的药物递送。Angiopep-2 (Ang, TFFYGGSRGKRNNFKTEEYC) 是靶向LRP1的最具代表性的肽, 其序列是基于阿普罗汀及其他具有Kunitz型结构域的LRP1结合蛋白的序列比对而设计的^[112]。Angiopep-2能够促进药物穿越BBB并靶向胶质瘤细胞, 已被广泛应用于治疗GBM^[113]。Zou等^[114]研究开发了一种高效封装CRISPR/Cas9的纳米胶囊, 通过系统给药实现对GBM的非侵入性靶向治疗。这些纳米胶囊采用原位聚合方法, 在GSH降解的聚合物壳内封装Cas9/sgRNA RNP, 通过使用Angiopep-2肽作为靶向配体, Cas9 RNP纳米胶囊在GBM小鼠模型中展现了明显的肿瘤靶向能力(11.8% ID·g^-1) 和高效的PLK1基因编辑效果(Sanger测序显示28.6%的插入/缺失率), 将携带肿瘤小鼠的中位生存时间从24天延长至68天。此外, Angiopep-2肽的修饰使纳米颗粒能够高效穿越BBB并在脑肿瘤中积累(12.9% ID·g^-1)。在GBM小鼠模型中, Cas9介导的PLK1基因敲除有效抑制了肿瘤生长, 将小鼠的中位生存时间从18天(PBS处理) 延长至40天, 同时几乎没有不良反应^[115]。

3.3.2　载体蛋白介导的胞吞转运

CMT是通过特定的膜载体蛋白在逆浓度梯度中将药物运过BBB^[116]。BBB内皮细胞含有多种特异性转运蛋白, 如葡萄糖转运蛋白(glucose transporter type 1, GLUT1)、氨基酸转运蛋白(L-type amino acid transporter 1, LAT1)、核苷转运蛋白等神经递质、维生素和脂肪酸的转运蛋白。这些转运蛋白通常位于内皮细胞的顶端和基底侧膜上。药物被设计成类似于这些天然底物(如葡萄糖、氨基酸等) 的分子, 这样转运蛋白能够识别并结合这些药物分子。当转运蛋白识别并结合药物分子以后, 其构象会发生变化, 能够将药物从血液一侧转运到脑内一侧。由此可见改变纳米颗粒上的某些转运蛋白或其类似物是增加纳米颗粒BBB渗透性的有效方法。

GLUT1是一种己糖转运蛋白。Na离子浓度差异驱动D-葡萄糖、L-抗坏血酸及其衍生物通过GLUT1从血液转运到大脑^{[117, 118]}。Zhou等^[119]开发了一种半乳糖修饰的两亲性聚合物胶束系统包载siRNA药物(Gal-NP@siRNA), 该系统通过血糖控制的GLUT1介导的转运高效穿越BBB。研究表明, Gal-NP@siRNA系统在AD小鼠模型中显著降低了BACE1的表达, 减少了淀粉样斑块, 并改善了认知功能, 同时未显示出显著的不良反应, 展示了其在中枢神经系统疾病治疗中的巨大潜力。

3.3.3　吸附介导的胞吞转运

AMT提供了一种通过细胞吸附和胞吞作用将物质跨越BBB向大脑递送药物的方法^[100]。其相关机制是带正电的蛋白质、肽或分子与BBB细胞内腔侧的负电糖萼和细胞膜发生静电相互作用, 触发内吞作用, 形成跨胞吞噬小泡。这些小泡移动到BBB细胞的外腔膜, 融合并释放分子进入大脑^[120]。

富含阳离子氨基酸(如精氨酸或赖氨酸) 的CPPs可通过与内皮细胞的静电吸附增强BBB的通透性^[121]。Yao等^[122]研究了一种新型基因递送系统，用于BBB治疗胶质瘤。该系统基于树枝状聚赖氨酸(dendrigraft poly-L-lysines, DGL) 和PEG结合的细胞穿透肽(LIM kinase 2 nucleolar translocation peptide, LNTP) LIM激酶2核仁转运信号序列。基因治疗载体(DGL-PEG-LNTP) 封装了编码生长抑制因子基因的pDNA。研究结果表明, 该递送系统能够有效穿越BBB, 增强细胞摄取, 并促进肿瘤细胞内的基因表达。LNTP修饰的纳米颗粒(DGL-PEG-LNTP/DNA) 在胶质瘤小鼠模型中显示出显著的肿瘤细胞凋亡效果, 并且与其他治疗相比, 显著延长了胶质瘤小鼠的中位生存时间。Chen等^[98]设计了用于将CRISPR/Cas9基因组编辑器递送到大脑的脂质体模板水凝胶纳米颗粒(liposome-templated hydrogel nanoparticles, LHNPs)。LHNPs修饰了iRGD和mHph3 CPPs以增强细胞内递送效率, 使其在脑肿瘤中的积累比未修饰的LHNP高出2.6倍。但同时利用CPPs进行修饰可能会促进药物在全身给药后被不适当的细胞吸收, 从而产生不良反应。此外, CPPs的正电荷还可能加速纳米颗粒在血液循环中的清除。因此, 应谨慎考虑在全身给药中应用CPPs。

4　核酸药物的脑部递送策略及其应用

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核酸药物的强负电荷和对核酸酶降解的易感性限制了它们被局灶细胞吸收, 临床应用面临稳定性差、靶向性弱、难以跨越体内屏障等难题。不同类型的核酸具有不同的分子结构和分子质量, 因此需要不同的装载策略: 小的核酸药物, 如siRNA、miRNA和ASOs, 可以通过核酸化学结构修饰来提高其在体内的稳定性和靶向性, 并与配体分子偶联以促进其在病灶内的积累, 提高递送效率, 同时降低药物的免疫原性; 而分子质量相对较大的核酸药物, 如mRNA和pDNA, 难以进行化学修饰, 易于通过具有较大空间的载体进行装载和体内递送, 目前应用较多的递送载体有病毒载体、聚合物纳米载体、脂质纳米颗粒、无机纳米载体、蛋白载体和外泌体等。这些载体不仅帮助药物到达病灶, 还促进核酸类药物实现更高效的内体逃逸, 从而在体内更好地发挥作用。向大脑递送核酸的理想载体应具有生物相容性、稳定性和有效的细胞摄取等特殊特性。穿过BBB的扩散主要取决于BBB两侧药物的亲脂性、表面电荷、相对分子质量和浓度梯度。不同载体在核酸药物脑部递送方面的应用情况如表2^{[52, 55, 85, 88, 89, 98, 103, 106, 110, 114, 119, 122-141]}所示。

4.1　病毒载体

病毒载体, 包括腺相关病毒(adeno-associated virus, AAV)、腺病毒、慢病毒、逆转录病毒和单纯疱疹病毒, 已被开发并用于中枢神经系统疾病的临床前和临床基因治疗。研究发现, 病毒可以通过不同的机制穿越BBB (图3A), 如AAV通过与神经血管内皮细胞上的表面蛋白结合进入脑实质, 触发受体介导的转吞作用, 可以转导神经元、星形胶质细胞、少突胶质细胞和内皮细胞^[142-144], 并且能够在神经细胞中长期表达。人类免疫缺陷病毒(human immunodeficiency virus, HIV) 可以感染单核细胞并利用其作为特洛伊木马来穿越BBB^[145]。BBB外感觉和运动神经元是狂犬病毒等病毒的另一入口, 他们结合神经肌肉连接处的神经元并利用轴突运输系统进行逆行运输^[92]。基于此已经开发了几种基于病毒颗粒递送治疗性核酸的方法。2012年欧洲药品管理局(European Medicines Agency, EMA) 批准了首个基于AAV方法的基因治疗产品Glybera, 展示了AAV载体的理想安全性和巨大应用潜力。而第一个成功治疗中枢神经系统疾病的基于AAV的基因疗法是onasemnogene abeparvovec (Zolgensma), 该疗法已被FDA批准用于治疗年龄小于2岁的SMA儿童患者(NCT03955679)^[146]。

基于病毒的载体目前主要用于基因治疗, 并且通常依赖于侵入性递送(如IT)。这些方法存在一些问题, 包括免疫反应可能导致的药物降效、病毒载体的安全性风险及对正常细胞的影响、传递效率不稳定、生产和规模化困难, 以及可能引发基因编辑问题^[147]。

4.2　核酸药物结构改造

通过对核酸药物进行结构改造, 借助化学修饰手段将亲脂性分子引入核酸主链, 或采用配体偶联方式将多肽小分子配体等与核酸主链相连(图3B), 可使得到的核酸药物具有明确且单一的分子结构, 显著增强其稳定性, 并有效降低批次间差异^[99]。同时通过这些改造策略提升药物的细胞摄取效率及在特定靶组织或细胞的富集能力, 有利于核酸药物的临床转化^{[148, 149]}。

由于小核酸药物通常带有负电荷且极性较强, 在穿膜或跨越BBB时面临巨大挑战, 通过引入亲脂性基团可改善核酸药物的脂溶性, 从而在肝外组织(包括中枢神经系统) 实现更有效的递送^{[150, 151]}。例如, Brown等^[126]研究发现, 2′-O-十六烷基(C₁₆) 修饰的siRNA可在啮齿类和非人灵长类动物中实现持续且高效的基因沉默。在AD小鼠模型中的进一步研究表明, C₁₆-siRNA能靶向淀粉样前体蛋白, 有效改善实验动物的行为及生理缺陷。除十六烷基外, 胆固醇的环状结构也常用于核酸修饰, 并被认为较直链脂肪酸更具亲脂性。Nagata等^[123]将胆固醇或α-生育酚偶联于DNA/RNA异源双链寡核苷酸(heteroduplex oligonucleotides, HDO) 后, 通过静脉或皮下给药可使其更容易跨越BBB, 在中枢神经系统中广泛分布并实现对靶基因高达90%的抑制率, 大大优于传统单链ASOs。

在提升细胞摄取与逃逸效率方面, CPPs带有正电荷或疏水性基团, 可增强药物与细胞膜的相互作用并促进内吞, 部分CPP还能在内体酸化环境中破坏或改变内体膜结构, 实现内体/溶酶体逃逸, 有效避免药物降解。与此同时, 若偶联分子能够识别并结合BBB上高表达的受体, 则可通过RMT将药物递送至中枢神经系统。Dastpeyman等^[124]将CPP HA2-ApoE (131~150) 与抗SMA药物诺西那生(nusinersen) 偶联, 通过HA2-ApoE与LDLR的相互作用触发RMT, 实现脑部递送, 在SMN2转基因小鼠模型中, 这种偶联物通过系统给药显著增加了大脑和脊髓中完整SMN2蛋白的水平, 表明了其高效的内体逃逸及BBB跨越能力。

此外, 叶酸(folic acid, FA) 修饰是典型的小分子配体修饰方式之一, 它在早期神经元发育和分化中起着重要作用, 包括胶质母细胞瘤在内的各种癌细胞类型, 都表达高水平的叶酸受体。通过内吞作用辅助药物穿越细胞膜, 并与肿瘤细胞或BBB上的叶酸受体结合, 叶酸修饰可实现核酸药物的靶向富集。Lee等^[125]构建了含有抗miR-21锁核酸(locked nucleic acid, LNA) 序列的多价叶酸, 三向结(three-way-junction, 3WJ) RNA纳米粒子, 递送抗miR-21 LNA到胶质母细胞瘤细胞。通过静脉注射FA-3WJ-LNA-miR21, 有效地降低了miR-21的表达, 从而恢复了肿瘤抑制因子PTEN和PDCD4的功能, 使得胶质母细胞瘤细胞凋亡和肿瘤生长抑制, 显著提高了小鼠的总体生存率。

综上可见, 化学修饰或配体偶联后的核酸药物不仅具备结构可控、成分单一、亲脂性可调等优势, 也能更好地应对快速肾脏清除及内吞体/溶酶体逃逸等挑战; 若再结合BBB跨越策略或特异性配体靶向机制, 则有望进一步提高核酸药物在多种疾病模型中的治疗效率和安全性, 为后续临床转化奠定基础。

4.3　脂质纳米颗粒

脂质类是核酸药物递送中应用最广泛的一类递送系统, 包括脂质体(liposome)、脂质体复合物(lipoplex, LP)、脂质纳米颗粒(lipid nanoparticle, LNP) 等, 目前研究中用得较多的为含有可离子化脂质的LNP^[152]。LNP一般包括可电离的阳离子磷脂(ionizable lipids)、中性辅助磷脂(helper lipids)、胆固醇、聚乙二醇修饰的磷脂(PEGylated lipid) 等4种主要成分(图3C)^[153]。

脂质纳米颗粒由于其尺寸小并且具有亲脂性而容易被大脑吸收, 这同时也延长了它们在血液中的循环时间并且更容易通过BBB运输^[154]。然而当静脉注射LNP时, 血流中吸附在纳米颗粒表面的血清蛋白冠通过ApoE受体介导的摄取会将其靶向肝脏^{[155, 156]}。针对这一问题, 研究人员通过对LNP进行修饰及设计相关的脂类纳米颗粒系统, 来改善其对BBB的递送^[127]。例如Su等^[127]制备了DPMT@PEI/miR-195纳米脂质体, 通过P-氨基苯基-α-D-吡喃甘露糖苷(P-aminophenyl-alpha-D-mannopyranoside, MAN) 和阳离子细胞穿透肽(cell-penetrating peptide, TAT) 双重修饰进行工程改造, 该脂质体表现出增强的BBB和细胞膜穿透能力。在APP/PS1小鼠模型中, 与多奈哌齐和阿杜那单抗相比, 通过尾静脉注射给药的DPMT@PEI/miR-195显示出更好的治疗效果, 能有效改善认知功能, 并减轻Aβ、tau蛋白过度磷酸化和小胶质细胞极化。

目前研究人员发现通过局部或鼻内给药的LNP能成功转染特定的细胞类型, 例如大脑内的小胶质细胞, 神经元和星形胶质细胞等^{[157, 158]}。Cohen等^[85]研究发现, 通过颅内注射(intracerebral injection, IC) 透明质酸功能化的LNP能成功帮助将siRNA递送至小鼠神经胶质瘤。Palanki等^[159]使用可电离脂质纳米颗粒作为递送平台, 通过脑室内注射, 筛选出一种在围产期大脑中具有比FDA批准的行业标准LNP更强的功能性mRNA递送能力的LNP配方(C3 LNP), 并通过进一步的优化使其能够改善新生小鼠大脑中溶酶体贮积病的生化表型。Ralvenius等^[128]优化改进了一种新型脂质纳米颗粒—先导小胶质细胞LNP, 腹腔注射和局部脑室内注射的方式都使其能够安全、高效靶向小胶质细胞, LNP介导的靶向PU.1转录因子(已知的AD风险位点) 的siRNA递送成功地降低了人诱导多能干细胞来源的小胶质样细胞(induced microglia-like cells, iMGL) 中的PU.1水平, 并减少了注射LPS的小鼠和CK-p25小鼠的神经炎症。但是像脑室内注射这些局部给药的方式往往是具有侵入性的, 因此现在一些研究也采用与非侵入性方法结合, 实现药物成功穿越BBB, 治疗中枢神经系统相关疾病。Zhao等^[129]设计一种新型复合物MB-shBirc5-lipo-NGR, 结合FUS, 可诱导非侵入性、可逆、局部BBB通透性, 通过实验证明了FUS辅助的MB-shBirc5-lipo-NGR可以打开BBB并抑制胶质瘤生长。

LNP具有亲水和疏水区域, 并且表面电荷可调, 使其能够封装各种理化性质不同的药物。这种多功能性允许在同一个纳米颗粒中共同封装不同类型的药物。在设计LNP时, 研究者不断优化脂质的化学结构, 以提高其在体内的稳定性和靶向性^[160]。例如, 通过改变脂质尾部的饱和度和连接子的化学结构, 可以调节LNP的流动性和内体逃逸能力^[161]。此外, 研究人员也在探索如何通过LNP表面修饰来提高其靶向特定细胞或组织的能力, 以及如何通过控制LNP的粒径和表面电荷来优化其在体内的生物分布和清除率。相比于病毒载体, 使用LNP递送核酸药物时可以有效规避感染、致癌及免疫原性等风险, 能有效保护核酸药物免受降解。同时通过添加辅料, LNP可以协助免疫激活和潜在的免疫应答, 进一步增强免疫效果。鉴于LNP在安全性和有效性方面的优势, 它们已成为药物递送和疫苗设计中的首选载体之一。并且LNP的安全性和有效性已得到了监管机构的广泛认可, 目前已成为FDA批准最多的药物载体类别^[99]。

尽管有这些优势, LNP仍面临一些挑战, 如容易被网状内皮系统快速吸收, 导致其在肝脏和脾脏中积累, 这不仅减少了核酸药物的可用性, 还增加了这些器官的负担。此外, LNP从内体和溶酶体中逃逸的能力有限, 严重影响了核酸药物的转染效率, 这也是当前LNP开发的关键关注点。

4.4　聚合物纳米载体

聚合物纳米颗粒(polymeric nanoparticles, PNPs) 是一种尺寸在1~1 000 nm的纳米材料^[148]。通常有纳米胶囊和纳米球两种形态结构, 纳米胶囊是一种囊状结构, 具有被聚合物膜或壳包裹的空腔, 可以控制其中的药物释放; 与纳米胶囊不同, 纳米球是一种固体颗粒系统, 药物或被包裹在构成纳米球的聚合物基质中, 或被吸附在颗粒表面(图3D)^[162]。纳米球的设计可以使药物从基质或表面逐渐释放出来, 从而实现药物的持续释放^[163]。在递送核酸药物的应用中, 聚合物纳米颗粒发挥了重要作用。它们通常是聚合物和核酸药物通常通过静电、疏水或氢键相互作用而形成的纳米颗粒。可以被分为多聚物、聚合体和树枝状大分子等几个亚类^[164]。

多聚物包含阳离子聚合物, 通过静电相互作用与核酸结合并凝聚成小而紧密的结构, 该凝聚过程是由熵驱动的, 当阳离子聚合物与核酸混合时, 多聚物自发产生。此方法可以产生表面带正电荷的颗粒, 并更好地将核酸凝聚成更小尺寸的纳米颗粒。该策略可以提高核酸药物的稳定性和细胞摄取效率。其中PEI具有高密度的正电荷, 能够实现高效的核酸药物装载及通过质子海绵效应逃逸内体/溶酶体^[99]。Rytblat等^[165]通过PEI的静电吸附增强了抗miR-125b ASO在胶质瘤细胞中的摄取和内体逃逸。MiR-125b在GBM细胞中的表达上调, 并通过促进细胞增殖和抑制凋亡, 在GBM细胞中发挥致癌效应。Sheikh等^[166]的研究显示, 聚赖氨酸修饰的PEI (PLys-PEI) 和PEI相比, 明显提高了核酸药物的转染效率, 同时降低了PNPs的毒性。通过将PLys-PEI与血管内皮生长因子(vascular endothelial growth factor, VEGF) pDNA相结合, 借助立体定位引导实施脑内注射, 发现负载有VEGF pDNA的PNP能够在神经元中实现高效转染, 显示出其在PD基因治疗领域的潜力。

聚合体是由两亲性嵌合共聚物制成的聚合物囊泡, 通过疏水效应自组装形成具有亲水核心和疏水涂层的纳米球, 具有亲水性的各种核酸(例如pDNA, ASO和siRNA) 可以被有效地封装到聚合物囊泡内部的水核中。例如Pangburn等^[167]发现由聚(1, 2-丁二烯)-b-聚(环氧乙烷) 嵌段共聚物自组装形成的聚合体, 可以用作siRNA的递送系统, 包封率约为51%。

树枝状大分子是由通过迭代的反应步骤, 在每次生长中增加分支, 形成具有树枝状结构的聚合物, 包含内核、重复单元和表面功能化3个部分。随着支化程度的增加, 最终形成封闭的三维球体, 内藏空腔结构, 表面可以通过将不同分子与反应性末端基团偶联来实现功能化。最常见的树枝状大分子有聚酰胺-胺(polyamidoamine, PAMAM)、聚赖氨酸[poly(L-lysine), PLL]、聚丙烯亚胺[poly(propylene imine), PPI] 等。Kim等^[168]使用可生物降解的PAMAM树枝状聚合物e-PAM-R包载编码高迁移率蛋白(high mobility group box protein 1, HMGB1) 的siRNA, 发现经鼻递送的HMGB1 siRNA能通过下调HMGB1表达, 抑制缺血后大鼠脑梗死体积, 并有助于神经和行为缺陷的恢复。Cai等^[132]设计了一种基于树突状结构DGL的siRNA和D肽(D peptide, Dp) 负载的纳米颗粒, 能够靶向并穿透BBB, 进入脑实质, 并在AD病变处积累。通过特异性靶向BBB上的转铁蛋白受体的T7肽和可酸裂解的长聚乙二醇, 实现高内化和有效的转胞吞作用。专门针对患病神经元的Tet1通过短PEG修饰为DGL, 驱动纳米颗粒到AD病灶并释放药物, 最终抑制了Aβ的产生, 缓解了Aβ斑块和tau蛋白磷酸化(p-tau) 缠结诱导的神经毒性, 显著提高了AD小鼠的认知能力。

目前研究发现通过BBB效率高度依赖于PNPs的物理化学特性。聚合物纳米颗粒提供了一种可调控的药物释放和降解特性, 通过改变其大小和表面电荷, 或者通过使用特定的修饰和靶向基团可以改善PNPs的血液循环时间、生物分布和穿透BBB的能力, 进一步增强其递送效率和细胞摄取率^[169]。

4.5　外泌体

近年来, 外泌体在核酸药物递送领域引起了广泛关注, 其独特的内源性生物学特性为多种治疗性核酸(如siRNA、miRNA、mRNA和ASO等) 的精准、高效运输提供了新的策略^{[170, 171]}。外泌体是一种异质性的内源细胞外囊泡(extracellular vesicles, EVs), 几乎可以由所有的细胞产生, 直径约为30~150 nm, 具有脂质双层膜结构, 膜上附着有黏附分子间质, 主要组织相容性复合体和多种蛋白质(四跨膜蛋、热休克蛋白、免疫调节蛋白等), 内腔含有多种多肽、核酸、氨基酸和代谢物等(图3E)^{[172, 173]}。作为细胞间通讯的关键参与者, 外泌体既可通过表面配体直接接触并激活受体细胞, 也可通过内吞作用将核酸、蛋白质和脂质在内的多种生物活性分子从供体细胞转移到受体细胞, 发挥信号转导作用^[174]。

外泌体具有良好的生物相容性和较低的免疫原性, 能够在细胞间传递核酸、蛋白等信号分子。在跨越BBB和实现脑靶向递送方面, 外泌体展现出了独特的优势, 其表面配体与大脑内皮细胞受体之间的相互作用被认为是外泌体脑靶向的主要机制。研究表明, 源自内皮细胞和树突状细胞的外泌体能够携带小分子药物和siRNA穿过BBB^{[136, 175]}。在小鼠卒中模型中, 与间充质干细胞衍生的EVs相比, 神经干细胞衍生的EVs显示出更高的脑靶向效率^[176]。然而, 外泌体在实际应用中仍面临一些挑战, 其生产和纯化过程复杂、产量低且稳定性差, 限制了大规模的生产和应用; 体内靶向性不够明确, 递送效率仍然有限, 影响了其治疗效果的发挥^[177]。

为了增强外泌体递送能力并克服其固有局限, 研究人员基于仿生理念, 将多种工程技术与外泌体的天然生物学特性融合, 开发工程化外泌体^[178]。通过基因工程和纳米工程技术等实现外泌体规模化生产和高效纯化, 并引入促稳定成分和特殊包覆技术增强其稳定性, 防止内部大分子荷载的降解或失活。同时, 表面修饰特定配体, 提高靶向性, 确保药物或基因精准递送至目标细胞, 提升治疗效果并减少不良反应^[179]。例如Alvarez-Erviti等^[136]发现通过静脉注射经过神经元特异性RVG肽修饰低免疫刺激性的树突状细胞来源的外泌体, 成功将GAPDH siRNA特异性地递送至大脑中的神经元、小胶质细胞和少突胶质细胞, 实现了特异性基因敲低, 且重复注射不影响效果; RVG外泌体递送BACE1 siRNA可显著降低阿尔茨海默病治疗靶点BACE1的mRNA和蛋白水平, 减少Aβ_1-42水平, 且未引起明显免疫反应。

相关的外泌体递送系统主要通过内源性和外源性两种载药方式载带核酸药物(图3F)^[180]。其中内源性加载是指通过基因工程、代谢标记和外源导入等策略对亲本细胞进行特异性修饰, 从而使得这些细胞分泌的外泌体携载需要递送的药物。常用细胞转染的内源性方式将核酸药物加载到外泌体中, 其优点是保留了外泌体的生物功能及其完整性, 但它同时需要复杂的操作步骤, 实验周期较长, 制备成本较高。外源性加载则是在分离和收集外泌体后采用共孵育、电穿孔、超声处理、反复冻融、共挤出等技术, 在外泌体表面或者内腔修饰和装载所需药物或者靶向试剂^[180], 相对来说更具有灵活性, 并且可以通过针对特定靶标的定向性修饰来提高靶向性。然而, 外源性药物装载也可能会面临外泌体的完整性被破坏, 有未被捕获的药物需要额外的步骤来除去等问题。

将治疗性外泌体递送到靶细胞有被动靶向和主动靶向两种方式, 其中的被动靶向是指不同类型的天然外泌体可以黏附不同的细胞, 具有来源于供体细胞的天然靶向能力。而主动靶向则利用各种技术方法通过外泌体表面工程实现外泌体的靶向递送^[173]。

外泌体在脑部核酸药物递送方面具有极大的潜力, 虽然目前像脂质体、聚合物纳米颗粒等制剂都可以利用其纳米尺寸跨越BBB来实现核酸药物的脑靶向递送, 但会存在血液毒性和机体内蓄积的问题; 而本身就具有穿透BBB的内源性外泌体具有更低的毒性和免疫原性, 生物相容度高, 血液循环稳定性和组织选择性好, 并且可以对其进行复杂的工程化改造, 是一种具有发展潜力的脑部核酸药物递送体系。

5　展望与挑战

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核酸药物, 包括siRNA、miRNA、ASOs、mRNA、pDNA和CRISPR/Cas系统, 以其在基因水平上的精准调控, 革新了神经系统疾病的治疗思路, 也为更多健康问题的解决提供了新的机会。它们能够精确调控基因表达, 为神经退行性疾病、脑肿瘤和脑血管疾病等复杂疾病提供了新的治疗方法。随着生物医学技术的不断进步, 核酸药物已经成为21世纪生物技术革命的前沿, 它们不仅补充了传统药物, 还代表着生物治疗方式从“被动控制”向“主动干预”的范式转移: 通过精准调控基因表达, 直接干预疾病的发生和发展, 从根本上改变疾病的进程。核酸药物的成功应用有望从分子和细胞层面重塑疾病的病理机制, 为遗传性和后天性脑部疾病的治疗带来进一步突破。

然而, 要实现核酸药物的有效应用, 必须克服BBB这一生理障碍。尽管BBB作为大脑的生理防护系统能有效阻挡有害物质入侵, 但同时也限制了治疗性大分子药物的进入。为突破这一瓶颈, 科研人员开发了多种创新策略, 包括利用超声波和电穿孔等物理方法暂时性地增加BBB的通透性, 以及开发病毒载体、LNP、聚合物纳米颗粒和生物源性外泌体等多样化递送系统。这些递送系统各具特色, 展现了不同的优势, 也面临着各自的挑战: 病毒载体可能引发安全隐患, LNP可能诱发免疫反应, 而外泌体则存在规模化生产困难等问题。为了解决这些问题, 研究者不仅致力于优化现有递送系统的效率, 还不断创新, 通过生物工程手段对递送载体进行表面修饰和功能化设计, 以增强其在穿越BBB时的靶向性和生物相容性。例如, 表面修饰靶向分子或肽类, 可以帮助递送系统更有效地穿越BBB, 并将核酸药物递送到特定的脑区或细胞。同时, 还在开发多功能纳米载体, 这些载体集成了靶向、响应性释放和成像功能, 能够实现更精准的递送和实时监控。

这些技术的进步不仅为核酸药物的递送开辟了新途径, 也在重塑药物递送的未来, 推动中枢神经系统疾病治疗向精准医学迈进。核酸药物的应用标志着药物开发领域的革命性突破, 并体现了医学科学向跨学科融合和个性化治疗策略的重要转型。随着递送技术的不断优化和创新, 核酸药物有望在更广泛的医疗领域发挥作用, 推动疾病治疗范式从“控制症状”向“精准治愈”转变, 为人类健康开辟新的前沿。

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doi: 10.16438/j.0513-4870.2024-1196

接收时间：2024-12-04
首发时间：2026-09-20
出版时间：2025-08-12

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收稿日期：2024-12-04
修回日期：2025-04-02

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上海交通大学医学院, 上海 200025

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高小玲, Tel: 86-21-63846590-776580, E-mail: shellygao1@sjtu.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

Drug name	Condition	Developer	Type	Target	Delivery method	Phase	Year approved or clinicaltrials.gov ID
Nusinersen (Spinraza)	SMA	Ionis/Biogen	ASOs	SMN2	IT	Approved (FDA)	2016
Tofersen (Qalsody)	ALS	Ionis/Biogen	ASOs	SOD1	IT	Approved (FDA)	2023
Ulefnersen (ION363)	ALS	Ionis	ASOs	FUS	IT	Ⅲ	NCT04768972
Zilganersen (ION373)	Alexander disease	Ionis	ASOs	GFAP	IT	Ⅲ	NCT04849741
Tominersen (IONIS-HTTRx)	HD	Ionis/Roche	ASOs	HTT	IT	Ⅱ	NCT05868551, NCT04000594
IONIS-MAPTRx (BIIB080)	AD	Ionis/Biogen	ASOs	MAPT	IT	Ⅱ	NCT03186989, NCT02835117
AMT-130	HD	UniQure Biopharma	Gene therapy (AAV vector)	HTT	Intra-striatal	Ⅰ/Ⅱ	NCT04120493
WVE-003	HD	Wave Life Sciences	siRNA	HTT	IT	Ⅰ/Ⅱ	NCT05032196
ALN-APP	Early-onset AD (EOAD)	Alnylam/Regeneron	siRNA	APP	IT	Ⅰ/Ⅱ	NCT05231785
STK-001	Dravet syndrome (DS)	Stoke Therapeutics	siRNA	SCN1A	IT	Ⅱ	NCT04740476
ION859 (BIIB094)	PD	Ionis/Biogen	ASOs	LRRK2	IT	Ⅰ/Ⅱ	NCT03976349
ION582	Angelman syndrome (AS)	Ionis/Biogen	ASOs	UBE3A-ATS	IT	Ⅰ/Ⅱ	NCT05127226
ION464 (BIIB101)	Multiple system atrophy (MSA)	Ionis	ASOs	SNCA	IT	Ⅰ/Ⅱ	NCT04165486

Drug name

Condition

Developer

Type

Target

Delivery method

Phase

Year approved or clinicaltrials.gov ID

Nusinersen
(Spinraza)

SMA

Ionis/Biogen

ASOs

SMN2

Approved
(FDA)

2016

Tofersen
(Qalsody)

ALS

Ionis/Biogen

ASOs

SOD1

Approved
(FDA)

2023

Ulefnersen
(ION363)

ALS

Ionis

ASOs

FUS

Ⅲ

NCT04768972

Zilganersen
(ION373)

Alexander disease

Ionis

ASOs

GFAP

Ⅲ

NCT04849741

Tominersen
(IONIS-HTTRx)

Ionis/Roche

ASOs

HTT

Ⅱ

NCT05868551,
NCT04000594

IONIS-MAPTRx
(BIIB080)

Ionis/Biogen

ASOs

MAPT

Ⅱ

NCT03186989,
NCT02835117

AMT-130

UniQure Biopharma

Gene therapy
(AAV vector)

HTT

Intra-striatal

Ⅰ/Ⅱ

NCT04120493

WVE-003

Wave Life Sciences

siRNA

HTT

Ⅰ/Ⅱ

NCT05032196

ALN-APP

Early-onset AD
(EOAD)

Alnylam/Regeneron

siRNA

APP

Ⅰ/Ⅱ

NCT05231785

STK-001

Dravet syndrome (DS)

Stoke Therapeutics

siRNA

SCN1A

Ⅱ

NCT04740476

ION859
(BIIB094)

Ionis/Biogen

ASOs

LRRK2

Ⅰ/Ⅱ

NCT03976349

ION582

Angelman syndrome (AS)

Ionis/Biogen

ASOs

UBE3A-ATS

Ⅰ/Ⅱ

NCT05127226

ION464
(BIIB101)

Multiple system atrophy (MSA)

Ionis

ASOs

SNCA

Ⅰ/Ⅱ

NCT04165486

Type	Composition	Drug type	Mechanism	Disease	Target	Administration	Result	Ref.
Nucleic acid drug conjugation	DNA/RNA heteroduplex with cholesterol conjugation at the 5′ end of RNA strand	ASOs	Cholesterol-mediated transcytosis (cholesterol)	CNS-related conditions	Malat1	Ⅳ	Knockdown of Malat1 RNA in neurons, astrocytes, and microglial cells by up to 90%	[123]
HA2, ApoE-derived peptides, conjugated to phosphorodiamidate morpholino oligomer (PMO) antisense oligonucleotides	ASOs	RMT (ApoE peptides)	SMA	SMN2	Ⅳ	Improved exon-7 inclusion activity in the CNS, increased pharmacological efficacy and uptake in neuronal cells	[124]
Folate (FA)-conjugated RNA, anti-miR-21 LNA	siRNA	RMT (FA)	GBM	miRNA-21	Ⅳ	Effective knock down of miR-21 in glioblastoma rescue of tumor suppressor genes (PTEN, PDCD4), induced apoptosis	[125]
2′-O-Hexadecyl (C₁₆) conjugated siRNA	siRNA	Bypass BBB (IT or ICV)	AD	APP & SOD1	IT or ICV	Effective and durable gene silencing in CNS and lungs, up to 90% knockdown in animal models	[126]
Lipid-based nanoparticles	Lipid mixture (DODAP, DSPC, cholesterol, PEG-ceramide) encapsulating siRNAs with RVG-9r peptide	siRNA	RMT (TFR) (RVG)	Machado-Joseph disease (MJD)	ATXN3	Ⅳ	Significant reduction in mutant ataxin-3, reduced neuropathology, improved motor behavior in mouse models	[55]
Hyaluronic acid (HA), Dlin-MC3-DMA, DSPC, cholesterol, PEGylated lipids, siPLK1	siRNA	Bypass BBB (CDE)	GBM	PLK1	IC	80% Reduction in PLK1 mRNA levels, increased cell death in glioma cells, prolonged survival in GBM-bearing mice	[85]
PLGA, DPPC, Liss Rhod PE, DSPE-PEG, MVL5	siRNA	Open BBB (MB-FUS)	Glioma, medulloblastoma	SMO	Ⅳ	10-Fold increase in siRNA delivery, significant SMO knockdown, tumor reduction, and cancer cell apoptosis	[88]
DOPE, DOTAP, DSPE-PEG-COOH, Tf, penetratin, and chitosan-pDNA complex	pDNA	RMT & AMT (TFR) Tf & cpp	Neurodegenerative diseases	Neurons in the brain	Ⅳ	Efficient transfection of brain neurons and significant accumulation in the brain	[106]
SPIONs, DOPA-PLys-PEG-RVG, Flu	CRISPR/Cas9 fluvastatin	RMT (TFR) (RVG)	AD	BACE1	Ⅳ	Significantly improved cognition in AD mice, reduced amyloid-β plaques, efficient gene editing and synergistic effects	[110]
PEI-based hydrogel core, DOTAP, cholesterol, DSPE-PEG2000-MAL, iRGD-conjugation	CRISPR/Cas9	RMT & open BBB (iRGDs & LEX)	Brain tumour	PLK1	Ⅳ	Efficient PLK1 gene knockout, significant tumor inhibition, prolonged survival in mouse models	[98]
DSPE-PEG, EPC cholesterol, PEI, MAN, TAT peptide	miRNA	CMT (Glut1) & AMT (MAN ligand & TAT peptide)	AD	APP, BACE1	Ⅳ	Reduced Aβ and tau levels, improved cognitive function in mouse models	[127]
Ionizable lipid, cholesterol, phospholipid, PEG-lipid	siRNA	Bypass BBB (local intracisternal injection, IC)	AD	PU.1	IC	Successfully reduced neuroinflammation and decreased PU.1 levels in microglia	[128]
DPPC, DSPE-PEG2000, DSPE-COOH-PEG2000, DC-cholesterol	shRNA	Open BBB (FUS-MB)	Glioma	Birc5 gene, CD13	Ⅳ	Tumor growth was significantly reduced, with increased apoptosis and extended survival observed in glioma rats treated with FUS-assisted MB-shBirc5-lipo-NGR	[129]
DOPA, DMPC, ApoE3	siRNA	RMT (LDLR) (ApoE3)	GBM	ATF5	Ⅳ	Effective siRNA delivery, increased apoptosis inhibited tumor growth	[130]
DPPC, DOTAP, cholesterol	mRNA	Bypass BBB (nose to brain)	Neurological disorders	Brain neurons	IN	Significant mRNA transfection and protein expression in brain regions like cortex, striatum, midbrain; minimal systemic exposure	[131]
DPH/TPGAS NPs (composed of two amphiphilic polymers, D-PEG-Phis and T-PA-G-SA	siRNA	RMT (CTGF receptors) (DAG peptide)	AD	NF-κB, Aβ, tau protein	Ⅳ	Reduced Aβ and tau, improved memory and cognition in AD mouse models	[52]
PNP-based nanoparticles	PBAE, PEGylated	mRNA or CRISPR/Cas9 with sgRNA	Open BBB (FUS-MB)	Brain tumors, neurodegenerative diseases	/	Ⅳ	Effective nucleic acid delivery and genome editing in astrocytes and neurons, precise targeting of the FUS-treated brain region	[89]
Galactose-modified PEG-b-P(Gu), siRNA, fluorinated polymer	siRNA	CMT (Glut1)	AD	BACE1	Ⅳ	Reduced Aβ plaques, improved cognitive function, and reduced tau protein phosphorylation in AD mouse models	[119]
DGL conjugated with PEG and LIMK2 NoLS peptide	pDNA	AMT (LIMK2 NoLS peptide)	Glioma	ING4 gene (tumor suppressor)	Ⅳ	Enhanced BBB penetration, increased gene expression in tumor cells, increased apoptosis, extended survival	[122]
PEG-modified DGL, T7 peptide, Tet1 peptide	siRNA and D-peptide (Dp)	RMT(TFR) (T7 peptide & Tet1 peptide)	AD	BACE1, Aβ, p-tau	Ⅳ	Reduced β-amyloid plaques and tau protein phosphorylation; improved cognitive function in AD mice	[132]
PEGylated dendrigraft poly-L-lysines (DGL)	Non-coding RNA plasmid	RMT (RVG29 peptide)	AD	BACE1, tau protein	Ⅳ	Reduced Aβ plaques and tau fibrils, improved cognitive function, reduced neurodegeneration in AD mice	[133]
Glutathione-sensitive polymer, agiopep-2, Cas9/sgRNA	CRISPR/Cas9 complex	RMT(LRP) (Angiopep-2)	GBM	PLK1	Ⅳ	High PLK1 gene editing efficiency (up to 38.1%) in brain tumor, extended survival in mice (68 vs 24 days)	[114]
PEGylated DGL modified with Aleuria aurantia lectin (AAL) and KLVFF peptides	siRNA and rapamycin	Bypass BBB (nose to brain)	AD	BACE1, Aβ	IN	Reduced Aβ deposition, improved cognition, and induced autophagy in AD mice	[134]
Disulfide-crosslinked polymer, Angiopep-2	siRNA	RMT (LRP) (Angiopep-2)	GBM	PLK1	Ⅳ	Efficient BBB penetration, high tumor targeting, significant inhibition of glioblastoma growth, prolonged survival in GBM-bearing mice, minimal off-target effects	[135]
Transferrin-coated chimeric polymersomes (CPs)	siRNA	RMT (TFR) (TF)	Brain metastatic breast cancer	PLK1	Ⅳ	Effective tumor inhibition, enhanced survival, and specific RNA interference targeting metastatic brain tumors with transferrin-coated polymersomes	[103]
Exosomes derived from dendritic cells, modified with Lamp2b-RVG peptide	siRNA	RMT (nAChR) (RVG)	AD	BACE1	Ⅳ	Specific silencing of target genes (BACE1), reduction in mRNA and protein levels, reduced β-amyloid accumulation	[136]
	Exosomes derived from HEK293T cells, functionalized with an anti-EGFR single-chain variable fragment (scFv) antibody	siRNA	RMT (EGFR) (scFv)	NSCLC	LPCAT1	Ⅳ	Effectively delivered siRNA across the BBB to brain metastasis, showing significant antitumor efficacy with minimal toxicity	[137]
EV-based nanoparticles	Exosomes derived from hepatocytes, modified with LAMP2b-RVG29	siRNA	RMT (nAChR) (RVG29)	HD	HTT	Ⅳ	Reduction of mHTT protein and aggregates, improved motor function, and reduced neuropathology	[138]
Exosomes derived from serum of MCAO rats after electroacupuncture treatment	miRNA	Bypass BBB (IC)	Ischemic stroke	HIF-1α, VEGF, Notch1	IC	Decreased infarct volume, reduced pathological damage, increased angiogenic factor expression	[139]
Exosomes derived from HEK293 cell, decorated with T7-peptide	ASOs	RMT (TFR) (T7-peptide)	GBM	Anti-miR-21	Ⅳ	Significant reduction in tumor size, enhanced delivery to brain, higher uptake by glioblastoma cells	[140]
Macrophage-derived exosomes functionalized with panobinostat and PPM1D-siRNA	siRNA	RMT (cyclic RGD peptide, cRGD)	Gliomas	PPM1D	Ⅳ	Inhibited DIPG tumor growth, increased apoptosis, prolonged survival in DIPG-bearing mice	[141]

Type

Composition

Drug type

Mechanism

Disease

Target

Administration

Result

Ref.

Nucleic acid drug conjugation

DNA/RNA heteroduplex with cholesterol conjugation at the 5′ end of RNA strand

ASOs

Cholesterol-mediated transcytosis
(cholesterol)

CNS-related conditions

Malat1

Ⅳ

Knockdown of Malat1 RNA in neurons, astrocytes, and microglial cells by up to 90%

[123]

HA2, ApoE-derived peptides, conjugated to phosphorodiamidate morpholino oligomer (PMO) antisense oligonucleotides

ASOs

RMT (ApoE peptides)

SMA

SMN2

Ⅳ

Improved exon-7 inclusion activity in the CNS, increased pharmacological efficacy and uptake in neuronal cells

[124]

Folate (FA)-conjugated RNA, anti-miR-21 LNA

siRNA

RMT (FA)

GBM

miRNA-21

Ⅳ

Effective knock down of miR-21 in glioblastoma rescue of tumor suppressor genes (PTEN, PDCD4), induced apoptosis

[125]

2′-O-Hexadecyl (C₁₆) conjugated siRNA

siRNA

Bypass BBB (IT or ICV)

APP & SOD1

IT or ICV

Effective and durable gene silencing in CNS and lungs, up to 90% knockdown in animal models

[126]

Lipid-based nanoparticles

Lipid mixture (DODAP, DSPC, cholesterol, PEG-ceramide) encapsulating siRNAs with RVG-9r peptide

siRNA

RMT (TFR)
(RVG)

Machado-Joseph disease (MJD)

ATXN3

Ⅳ

Significant reduction in mutant ataxin-3, reduced neuropathology, improved motor behavior in mouse models

[55]

Hyaluronic acid (HA), Dlin-MC3-DMA, DSPC, cholesterol, PEGylated lipids, siPLK1

siRNA

Bypass BBB (CDE)

GBM

PLK1

80% Reduction in PLK1 mRNA levels, increased cell death in glioma cells, prolonged survival in GBM-bearing mice

[85]

PLGA, DPPC, Liss Rhod PE, DSPE-PEG, MVL5

siRNA

Open BBB
(MB-FUS)

Glioma, medulloblastoma

SMO

Ⅳ

10-Fold increase in siRNA delivery, significant SMO knockdown, tumor reduction, and cancer cell apoptosis

[88]

DOPE, DOTAP, DSPE-PEG-COOH, Tf, penetratin, and chitosan-pDNA complex

pDNA

RMT & AMT (TFR)
Tf & cpp

Neurodegenerative diseases

Neurons in the brain

Ⅳ

Efficient transfection of brain neurons and significant accumulation in the brain

[106]

SPIONs, DOPA-PLys-PEG-RVG, Flu

CRISPR/Cas9 fluvastatin

RMT (TFR)
(RVG)

BACE1

Ⅳ

Significantly improved cognition in AD mice, reduced amyloid-β plaques, efficient gene editing and synergistic effects

[110]

PEI-based hydrogel core, DOTAP, cholesterol, DSPE-PEG2000-MAL, iRGD-conjugation

CRISPR/Cas9

RMT & open BBB
(iRGDs & LEX)

Brain tumour

PLK1

Ⅳ

Efficient PLK1 gene knockout, significant tumor inhibition, prolonged survival in mouse models

[98]

DSPE-PEG, EPC cholesterol, PEI, MAN, TAT peptide

miRNA

CMT (Glut1) & AMT (MAN ligand & TAT peptide)

APP, BACE1

Ⅳ

Reduced Aβ and tau levels, improved cognitive function in mouse models

[127]

Ionizable lipid, cholesterol, phospholipid, PEG-lipid

siRNA

Bypass BBB
(local intracisternal injection, IC)

PU.1

Successfully reduced neuroinflammation and decreased PU.1 levels in microglia

[128]

DPPC, DSPE-PEG2000, DSPE-COOH-PEG2000, DC-cholesterol

shRNA

Open BBB
(FUS-MB)

Glioma

Birc5 gene, CD13

Ⅳ

Tumor growth was significantly reduced, with increased apoptosis and extended survival observed in glioma rats treated with FUS-assisted MB-shBirc5-lipo-NGR

[129]

DOPA, DMPC, ApoE3

siRNA

RMT (LDLR)
(ApoE3)

GBM

ATF5

Ⅳ

Effective siRNA delivery, increased apoptosis inhibited tumor growth

[130]

DPPC, DOTAP, cholesterol

mRNA

Bypass BBB
(nose to brain)

Neurological disorders

Brain neurons

Significant mRNA transfection and protein expression in brain regions like cortex, striatum, midbrain; minimal systemic exposure

[131]

DPH/TPGAS NPs (composed of two amphiphilic polymers, D-PEG-Phis and T-PA-G-SA

siRNA

RMT (CTGF receptors)
(DAG peptide)

NF-κB, Aβ, tau protein

Ⅳ

Reduced Aβ and tau, improved memory and cognition in AD mouse models

[52]

PNP-based nanoparticles

PBAE, PEGylated

mRNA or CRISPR/Cas9 with sgRNA

Open BBB
(FUS-MB)

Brain tumors, neurodegenerative diseases

Ⅳ

Effective nucleic acid delivery and genome editing in astrocytes and neurons, precise targeting of the FUS-treated brain region

[89]

Galactose-modified PEG-b-P(Gu), siRNA, fluorinated polymer

siRNA

CMT
(Glut1)

BACE1

Ⅳ

Reduced Aβ plaques, improved cognitive function, and reduced tau protein phosphorylation in AD mouse models

[119]

DGL conjugated with PEG and LIMK2 NoLS peptide

pDNA

AMT
(LIMK2 NoLS peptide)

Glioma

ING4 gene (tumor suppressor)

Ⅳ

Enhanced BBB penetration, increased gene expression in tumor cells, increased apoptosis, extended survival

[122]

PEG-modified DGL, T7 peptide, Tet1 peptide

siRNA and D-peptide (Dp)

RMT(TFR)
(T7 peptide & Tet1 peptide)

BACE1, Aβ, p-tau

Ⅳ

Reduced β-amyloid plaques and tau protein phosphorylation; improved cognitive function in AD mice

[132]

PEGylated dendrigraft poly-L-lysines (DGL)

Non-coding RNA plasmid

RMT
(RVG29 peptide)

BACE1, tau protein

Ⅳ

Reduced Aβ plaques and tau fibrils, improved cognitive function, reduced neurodegeneration in AD mice

[133]

Glutathione-sensitive polymer, agiopep-2, Cas9/sgRNA

CRISPR/Cas9 complex

RMT(LRP)
(Angiopep-2)

GBM

PLK1

Ⅳ

High PLK1 gene editing efficiency (up to 38.1%) in brain tumor, extended survival in mice (68 vs 24 days)

[114]

PEGylated DGL modified with Aleuria aurantia lectin (AAL) and KLVFF peptides

siRNA and rapamycin

Bypass BBB
(nose to brain)

BACE1, Aβ

Reduced Aβ deposition, improved cognition, and induced autophagy in AD mice

[134]

Disulfide-crosslinked polymer, Angiopep-2

siRNA

RMT (LRP)
(Angiopep-2)

GBM

PLK1

Ⅳ

Efficient BBB penetration, high tumor targeting, significant inhibition of glioblastoma growth, prolonged survival in GBM-bearing mice, minimal off-target effects

[135]

Transferrin-coated chimeric polymersomes (CPs)

siRNA

RMT (TFR)
(TF)

Brain metastatic breast cancer

PLK1

Ⅳ

Effective tumor inhibition, enhanced survival, and specific RNA interference targeting metastatic brain tumors with transferrin-coated polymersomes

[103]

Exosomes derived from dendritic cells, modified with Lamp2b-RVG peptide

siRNA

RMT (nAChR)
(RVG)

BACE1

Ⅳ

Specific silencing of target genes (BACE1), reduction in mRNA and protein levels, reduced β-amyloid accumulation

[136]

Exosomes derived from HEK293T cells, functionalized with an anti-EGFR single-chain variable fragment (scFv) antibody

siRNA

RMT (EGFR)
(scFv)

NSCLC

LPCAT1

Ⅳ

Effectively delivered siRNA across the BBB to brain metastasis, showing significant antitumor efficacy with minimal toxicity

[137]

EV-based nanoparticles

Exosomes derived from hepatocytes, modified with LAMP2b-RVG29

siRNA

RMT (nAChR)
(RVG29)

HTT

Ⅳ

Reduction of mHTT protein and aggregates, improved motor function, and reduced neuropathology

[138]

Exosomes derived from serum of MCAO rats after electroacupuncture treatment

miRNA

Bypass BBB
(IC)

Ischemic stroke

HIF-1α, VEGF, Notch1

Decreased infarct volume, reduced pathological damage, increased angiogenic factor expression

[139]

Exosomes derived from HEK293 cell, decorated with T7-peptide

ASOs

RMT (TFR)
(T7-peptide)

GBM

Anti-miR-21

Ⅳ

Significant reduction in tumor size, enhanced delivery to brain, higher uptake by glioblastoma cells

[140]

Macrophage-derived exosomes functionalized with panobinostat and PPM1D-siRNA

siRNA

RMT
(cyclic RGD peptide, cRGD)

Gliomas

PPM1D

Ⅳ

Inhibited DIPG tumor growth, increased apoptosis, prolonged survival in DIPG-bearing mice

[141]