RNA interference and antiviral therapy

Table 1 Strategies designed to inhibit HIV replication via RNA interference

Target gene	RNAi inducer (length)	Promoter	Vector	Cell type	Delivery methods	Inhib. of virus prod. (fold)	Reference
Viral Gene
LTR, Vif, Nef	siRNA (21 bp)/shRNA (19 bp)1	T7	Plasmid	Magi, PBLs	Transfection	> 20	[47]
Gag, Pol	siRNA (21 bp)	-	-	HOS.T4.CXCR4	Transfection	> 10	[48]
Gag, LTR	siRNA (23 bp)/dsRNA (21nt)2	-	-	U87-CD4+-CCR5+/CXCR4+, PBMC	Transfection	4	[51]
Gag, Env	dsRNA (441-531nt)3	-	-	COS, Hela-CD4+, PBMC, ACH2	Transfection	70	[52]
Tat + Rev	siRNA (21 bp)	-	-	293T, Jurkat, PBMC	Transfection	> 15	[54]
Rev (Tat)	siRNA (21 bp)5	Dual U6	Plasmid	293/EcR	Transfection	10000	[56]
Nef	dsRNA (556nt)3	-	-	MT4-T, U937	Transfection	2.5	[57]
Env	siRNA (20 bp)/shRNA (20 bp)4	U6	Plasmid, Lentivirus	COS, MT-4	Transfection /Transduction	> 10	[53]
Nef	shRNA (21 bp)	H1	Retrovirus	SupT1	Transduction	> 10	[58]
Gag, Pol, Int, Vpu	shRNA (21 bp)	H1	Lentivirus	293T, Magi, GHOST hi5, CEM-A, Molt-4, PBMC	Transduction	> 20	[61]
Cellular gene
Tsg101	siRNA (21 bp)	-	-	293T	Transfection	10-20	[63]
LEDGF/p75	siRNA (21 bp)	-	-	Hela	Transfection	NR	[64]
P-TEFb (CDK9/CyclinT1)	siRNA (21 bp)	-	-	Hela, Magi	Transfection	3-5	[65]
hRIP	siRNA (21 bp)	-	-	Hela, Jurkat, Macrophages	Transfection	-100	[66]
Emerin	siRNA (21 bp)	-	-	Hela, Macrophages	Transfection	> 10	[67]
LEDGF/p75, HRP2	siRNA (21 bp)	-	-	Hela-P4	Transfection	2-3	[68]
CXCR4	siRNA (21 bp)	-	-	HOS-CD4+, HOS-CD4+-CXCR4+/CCR5+	Transfection	3-5	[70]
Importin 7	siRNA (21 bp)	-	-	Hela, Macrophages	Transfection	-10	[69]
CXCR4+ CD4,
CXCR4+ CCR5	shRNA (19/21 bp)6	-	-	Magi-CXCR4/CCR5, PBMC	Transfection	> 15	[71]
CCR5	shRNA (19 bp)	U6	Lentivirus	Magi-CCR5, PBLs	Transduction	3-7	[34]
Combination of viral and cellular genes
Gag, CD4	siRNA (21 bp)	-	-	Magi-CCR5, Hela-CD4	Transfection	4-25	[50]
Tat, RT, NF-κB (p65)	siRNA (21 bp)	-	-	Magi, Jurkat	Transfection	5-500	[74]

The fold inhibition of virus production refers to the results obtained with the most potent siRNA/shRNA tested in a specific cell model. All siRNAs were prepared by chemical synthesis unless indicated otherwise. LTR: Long terminal repeat; PBLs: Peripheral blood lymphocytes; PBMC: Peripheral blood mononuclear cell; Pol: Polymerase; Env: Envelope; Tsg101: Tumor susceptibility gene 101; LEDGF/P75: Lens epithelium-derived growth factor/transcription co-activator p75; NR: Not reported; P-TEF: Positive transcription elongation factors; hRIP: Human Rev-interacting protein; HRP2: Hepatoma-derived growth factor related protein 2; RT: Reverse transcriptase; NF-κB: Nuclear factor-NF-κB.

¹shRNA expressed from a transfected plasmid under the control of a T7 promoter.

²dsRNA produced by in vitro T7 promoter-mediated transcription.

³dsRNA produced by in vitro SP6/T7 promoter-mediated transcription.

⁴shRNA and siRNA expressed from transfected plasmids under the control of one and two U6 promoters respectively, shRNA further stably expressed from a recombinant lentiviral vector driven by a U6 promoter.

⁵siRNA expressed from a transfected plasmid under the control of two U6 tandem promoters that drive the synthesis of each of the siRNA strand.

⁶shRNA produced by in vitro T7 promoter-mediated transcription.

Table 2 Strategies designed to inhibit HCV replication via RNA interference

Target gene	RNAi inducer (length)	Promoter	Vector	Model	Delivery methods	Inhib. of virus prod. (fold)	Reference
In vitro studies
Viral gene
5’-UTR	siRNA (21 bp)	-	-	5-2 cells (Huh-7)	Transfection	-6	[79]
	siRNA (21 bp)/shRNA(19 bp)1	U6	Plasmid	293T, Huh 7	Transfection	> 10	[78]
NS4B	siRNA (23 bp)	-	-	Huh-7.5	Transfection	-80	[80]
NS3, NS4B, NS5A, NS5B	siRNA (21 bp)	-	-	S1179I (Huh-7)	Transfection	-23	[81]
IRES, NS3, NS5B	siRNA (23bp)/shRNA (21 bp)2	Dual H1	Plasmid	Huh-7	Transfection	> 9	[82]
5’-UTR, C, NS4B, NS5A, NS5B	esiRNA (15-40 bp)3/shRNA (19bp)	H1	Mo-MuLV	Huh-7	Transfection / Transduction	-100	[88]
5’-UTR, C, NS3, NS5B	siRNA (21 bp)/shRNA (19 bp)4	U6	Plasmid/Lentivirus	Huh-7	Transfection / Transduction	-7	[83]
Cellular gene
Lα, PTB, eIF2Bγ, hVAP33	shRNA (19 bp)	U6	Plasmid/Adenovirus	Huh-7	Transfection / Transduction	-13	[91]
Cyp-A,B,C	shRNA (NR)	U6	Plasmid/Retrovirus	Huh-7	Transfection / Transduction	-10	[92]
Combination of viral and cellular genes
5’-UTR, 3’-UTR, PSMA7, HuR	shRNA (19-21 bp)	U6	Plasmid/Retrovirus	Huh-7	Transfection / Transduction	> 2	[89]
CD81, IRES, NS5B	shRNA (19-21 bp)	H1	Lentivirus	Huh-7	Transduction	> 32	[90]
In vivo studies
NS5B	siRNA (23 bp)	-	-	Mice	Hydrodynamic transfection	3	[84]
IRES	shRNA (19-25 bp)5	-	-	Mice	Hydrodynamic transfection	-50	[85]

The fold inhibition of virus production represents the most potent effect caused by a specific siRNA or combinatorial siRNAs. All siRNAs were prepared by chemical synthesis unless indicated otherwise. UTR: Untranslated region; NS: Non-structural; IRES: Internal ribosomal entry site; C: Core protein; esiRNA: Endoribonuclease-prepared siRNA; Mo-MuLV: Moloney murine leukemia virus; PTB: Polypyrimidine tract-binding protein; eIF2Bγ: Subunit gamma of human eukaryotic initiation factors 2B; hVAP-33: Human VAMP-associated protein of 33 kDa; Cyp: Cyclophilin; PSMA7: Proteasome a-subunit 7; HuR: Hu antigen R; N.R: not reported.

¹stem-loop- and tandem-type siRNA expressed from DNA-based vectors driven by one and two U6 promoters respectively.

²shRNA expressed from a transfected plasmid under the control of two H1 tandem promoters that drive the synthesis of each of the siRNA strand.

³esiRNA generated by in vitro T3/T7 promoter-mediated transcription.

⁴shRNA expressed from a transfected plasmid or a lentivirus vector respectively under the control of a U6 promoter. 5 shRNA generated by in vitro T7 promoter-mediated transcription.

Table 3 Strategies designed to inhibit HBV replication via RNA interference

Target gene	RNAi inducer (length)	Promoter	Vector	Model	Delivery methods	Inhib. of virus prod. (fold)	Reference
In vitro studies
C	siRNA (21 bp)	-	-	Huh-7, HepG2	Transfection	-4-5	[95]
	siRNA (19 bp)	-	-	HepAD38, HepAD79	Transfection	-50	[96]
C, X	shRNA (19 bp)	hH1	Plasmid	Huh-7, HepG2.2.15	Transfection	2-20	[97]
C, S, P, X, DR	shRNA (21-24 bp)	mU6	Plasmid	HepG2.2.15	Transfection	-2	[98]
	shRNA (21 bp)	hU6	Plasmid	HepG2	Transfection	> 30	[105]
S	shRNA (19 bp)	hH1	PFV, AAV	293T.HBs, HepG2.2.15	Transduction	4-9	[99]
In vivo studies
C, S, P, X	shRNA (25 bp)	hU6	Plasmid	Immunocompetent C57BL/6J mice, Immunocompromised NOD/SCID mice	Hydrodynamic transfection1	3-12	[100]
C, S	siRNA (21 bp)	-	-	Male NMRI mice	High-volume injection via tail vein1	-4	[101]
S	shRNA (19 bp)	hH1, hU6	Plasmid	BALB/c mice, HBsAg-transgenic FVB/N mice	Hydrodynamic transfection2	-9	[102]
P, S, X	shRNA (20 bp)	hH1	Plasmid	C57BL/6 HBV-transgenic mice	Hydrodynamic transfection1	19-99	[103]
P, S, X	shRNA (NR)	mU6	Adenovirus	HBV-transgenic mice	Hydrodynamic transfection	> 9	[104]

The fold inhibition of virus production refers to the results obtained with the most potent siRNA/shRNA. All siRNAs were prepared by chemical synthesis unless indicated otherwise. C: Core antigen; S: Surface antigen; P: Polymerase; X: X protein; DR: Direct repeat element; PFV: Prototype foamy virus; AAV: Adeno-associated virus; mU6: Mouse U6; hU6: Human U6; hH1: Human H1. 1shRNA expression plasmid/naked siRNA coinjected with the pHBV construct. 2shRNA expression plasmid simultaneously or subsequently injected with the pHBV/pSAg construct in BALB/c mice.

Table 4 Strategies designed to inhibit SARS-CoV replication via RNA interference

Target gene	RNAi inducer (length)	Promoter	Vector	Model	Delivery methods	Inhib. of virus prod. (fold)	Reference
In vitro studies Viral gene
Leader, TRS, 3’-UTR, S	siRNA (21 bp)	-	-	Vero E6	Transfection	9	[110]
N	shRNA (20 bp)	U6	Plasmid	293	Transfection	NR	[112]
E, M, N	siRNA (21 bp)	-	-	Vero E6	Transfection	> 4	[113]
P	shRNA (19 bp)	H1	Plasmid	Vero	Transfection	> 100	[114]
S	shRNA (22 bp)	U6	Plasmid	Vero E6, 293T	Transfection	-6	[115]
Rep	siRNA (21 bp)	-	-	FRhk-4	Transfection	> 12	[118]
Cellular gene
CHC	siRNA (25 bp)	-	-	HepG2, COS7	Transfection	-1	[116]
In vivo studies
S, NSP12	siRNA (21 bp)	-	-	BALB/C mouse, Rhesus macaque (Macaca mulatta)	i.t.1 and i.n.2 administration	3	[119]

The fold inhibition of virus production refers to the results obtained with the most potent siRNA/shRNA. All siRNAs were prepared by chemical synthesis unless indicated otherwise. TRS: Transcription-regulating sequence; UTR: Untranslated region; S: Spike protein; N: Nucleocapsid protein; NR: Not reported; E: Envelope protein; M: Membrane protein; P: RNA polymerase; Rep: Replicase; FRhk-4: Fatal Rhesus monkey kidney cells; CHC: Clathrin heavy chain; NSP: Non-structural protein; i.t.: Intratracheal; i.n.: Intranasal.

¹siRNA and target-sequence containing reporter plasmid co-administered intratracheally into mouse lungs in D5W or Infasurf solution;

²siRNA instilled intranasally to monkey in D5W solution with different dosing regimens.