Viral vectors expressing a single microRNA-based short-hairpin RNA result in potent gene silencing in vitro and in vivo

Osório, Luísa Alexandra Mendes; Gijsbers, Rik; Oliveras-Salvá, Marusela; Michiels, Annelies; Debyser, Zeger; Haute, Chris Van den; Baekelandt, Veerle

doi:10.1016/j.jbiotec.2013.11.004

Cited by 23 publications

(25 citation statements)

References 39 publications

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“…25 Nevertheless, efficient production of anti-HIV LVs expressing miR precursors has been reported. 30 In accordance with this, we did not observe significant differences when comparing the titers of control LVTH LV and pri-miR-encoding HBV-silencing LVs. The lack of an effect may be attributed to Drosha saturation in the producer cells and Rev response elementmediated export of unspliced and singly spliced genomic lentiviral RNA from the nucleus.…”

Section: Resultssupporting

confidence: 85%

“…Recombination events between repeated sequences in the lentiviral genome may occur during propagation of the recombinant virus, 32 and deletion of pri-miR sequence in the context of a tricistronic scaffold has been reported previously. 30,31 To assess whether recombination had occurred between equivalent sequences in the tricistronic pri-miR-31/5-8-9 cassette during LV transduction, genomic DNA extracted from LVTH mTTR miR-31/5-8-9-transduced Huh7 cells was analyzed by PCR ( Figure 1d). A single band of 493 bp, corresponding to the size of the mTTR pri-miR-31/5-8-9 cassette, was amplified from LVTH mTTR miR-31-/5-8-9 genomic DNA.…”

Section: Resultsmentioning

confidence: 99%

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Sustained inhibition of hepatitis B virus replication in vivo using RNAi-activating lentiviruses

et al. 2014

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Chronic infection with hepatitis B virus (HBV) puts individuals at high risk for complicating cirrhosis and liver cancer, but available treatment to counter the virus rarely eliminates infection. Although harnessing RNA interference (RNAi) to silence HBV genes has shown the potential, achieving efficient and durable silencing of viral genes remains an important goal. Here we report on the propagation of lentiviral vectors (LVs) that successfully deliver HBV-targeting RNAi activators to liver cells. Mono- and tricistronic artificial primary microRNAs (pri-miRs) derived from pri-miR-31, placed under transcriptional control of the liver-specific modified murine transthyretin (mTTR) promoter, caused efficient inhibition of HBV replication markers. The tricistronic cassette was capable of silencing a mutant viral target and the effects were observed without disrupting the function of an endogenous miR (miR-16). The mTTR promoter stably expressed a reporter transgene in mouse livers over a study period of 1 year. Good silencing of HBV genes, without evidence of toxicity, was demonstrated following intravenous injection of LVs into neonatal HBV transgenic mice. Collectively, these data indicate that LVs may achieve sustained inhibition of HBV replication that is appealing for their therapeutic use.

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Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Sustained inhibition of hepatitis B virus replication in vivo using RNAi-activating lentiviruses

et al. 2014

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“…For double transduction cells were first transduced with CDC50A vector, selected with hygromycin, and then transduced with the different ATP10B viral vectors. For knockdown, micro-RNA (mir) based short-hairpin lentiviral vectors were generated as described [82]. Viral vectors against 5 (human) to 7 (mouse) different target sequences were produced and validated for functionality.…”

Section: Viral Transductionsmentioning

confidence: 99%

Mutated ATP10B increases Parkinson’s disease risk by compromising lysosomal glucosylceramide export

et al. 2020

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Parkinson's disease (PD) is a progressive neurodegenerative brain disease presenting with a variety of motor and non-motor symptoms, loss of midbrain dopaminergic neurons in the substantia nigra pars compacta and the occurrence of α-synucleinpositive Lewy bodies in surviving neurons. Here, we performed whole exome sequencing in 52 early-onset PD patients and identified 3 carriers of compound heterozygous mutations in the ATP10B P4-type ATPase gene. Genetic screening of a Belgian PD and dementia with Lewy bodies (DLB) cohort identified 4 additional compound heterozygous mutation carriers (6/617 PD patients, 0.97%; 1/226 DLB patients, 0.44%). We established that ATP10B encodes a late endo-lysosomal lipid flippase that translocates the lipids glucosylceramide (GluCer) and phosphatidylcholine (PC) towards the cytosolic membrane leaflet. The PD associated ATP10B mutants are catalytically inactive and fail to provide cellular protection against the environmental PD risk factors rotenone and manganese. In isolated cortical neurons, loss of ATP10B leads to general lysosomal dysfunction and cell death. Impaired lysosomal functionality and integrity is well known to be implicated in PD pathology and linked to multiple causal PD genes and genetic risk factors. Our results indicate that recessive loss of function mutations in ATP10B increase risk for PD by disturbed lysosomal export of GluCer and PC. Both ATP10B and glucocerebrosidase 1, encoded by the PD risk gene GBA1, reduce lysosomal GluCer levels, emerging lysosomal GluCer accumulation as a potential PD driver.Keywords Parkinson's disease · Massive parallel sequencing · ATP10B P-type ATPase · Endo-lysosomal lipid flippase · Loss-of-function · Glucosylceramide Shaun Martin and Stefanie Smolders shared first authors and have contributed equally. Peter Vangheluwe and Christine Van Broeckhoven shared last and corresponding authors and have contributed equally.

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“…Prototype Foamy Virus chromatin binding segment of Gag 534-546 (PFV Gag 534-546 ) [52–54], Human Papilloma Virus serotype 5 E2 242-257 , Human Papilloma Virus serotype 8 E2 240-255 (HPV5 E2 242-257 and HPV8 E2 240-255 , respectively)[56–58] and Kaposi’s Sarcoma Herpes Virus Latency Associated Nuclear Antigen 1-31 (KSHV LANA 1-31 ) [55] were used to replace the PWWP domain, generating ΔN 93 -LEDGF, PFV Gag 534-546 -ΔN 93 -LEDGF, HPV5 E2 242-257 -ΔN 93 -LEDGF, HPV8 E2 240-255 -ΔN 93 -LEDGF and KSHV LANA 1-31 -ΔN 93 -LEDGF fusions, respectively. All above-mentioned LEDGF-hybrids were used to complement LEDGF/p75-depleted cells (HeLaP4 (LEDGF KD ) [60] and Nalm (LEDGF KO ) cells [67]) employing SIV-based lentiviral vectors. As a positive control, cells were complemented with WT LEDGF/p75 (referred to as LEDGF/p75 back complementation (LEDGF BC )).…”

Section: Resultsmentioning

confidence: 99%

“…A lentiviral vector carrying CMV promoter driving a Zeocin resistance gene and a LEDGF specific miRNA-based shRNA was described earlier [60] and used to generate stable LEDGF KD cells. All LEDGF/p75 hybrid expression constructs were cloned into the pGAE backbone and cloning steps sequence verified.…”

Section: Methodsmentioning

confidence: 99%

Towards a Safer, More Randomized Lentiviral Vector Integration Profile Exploring Artificial LEDGF Chimeras

et al. 2016

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The capacity to integrate transgenes into the host cell genome makes retroviral vectors an interesting tool for gene therapy. Although stable insertion resulted in successful correction of several monogenic disorders, it also accounts for insertional mutagenesis, a major setback in otherwise successful clinical gene therapy trials due to leukemia development in a subset of treated patients. Despite improvements in vector design, their use is still not risk-free. Lentiviral vector (LV) integration is directed into active transcription units by LEDGF/p75, a host-cell protein co-opted by the viral integrase. We engineered LEDGF/p75-based hybrid tethers in an effort to elicit a more random integration pattern to increase biosafety, and potentially reduce proto-oncogene activation. We therefore truncated LEDGF/p75 by deleting the N-terminal chromatin-reading PWWP-domain, and replaced this domain with alternative pan-chromatin binding peptides. Expression of these LEDGF-hybrids in LEDGF-depleted cells efficiently rescued LV transduction and resulted in LV integrations that distributed more randomly throughout the host-cell genome. In addition, when considering safe harbor criteria, LV integration sites for these LEDGF-hybrids distributed more safely compared to LEDGF/p75-mediated integration in wild-type cells. This approach should be broadly applicable to introduce therapeutic or suicide genes for cell therapy, such as patient-specific iPS cells.

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Viral vectors expressing a single microRNA-based short-hairpin RNA result in potent gene silencing in vitro and in vivo

Cited by 23 publications

References 39 publications

Sustained inhibition of hepatitis B virus replication in vivo using RNAi-activating lentiviruses

Sustained inhibition of hepatitis B virus replication in vivo using RNAi-activating lentiviruses

Mutated ATP10B increases Parkinson’s disease risk by compromising lysosomal glucosylceramide export

Towards a Safer, More Randomized Lentiviral Vector Integration Profile Exploring Artificial LEDGF Chimeras

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