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Micro-RNA based hairpins targeting conserved regions of HIV-1 for enhanced viral gene silencing

Title
Micro-RNA based hairpins targeting conserved regions of HIV-1 for enhanced viral gene silencing
Other Titles
HIV-1 치료를 위한 최적의 micro RNA 개발 및 항바이러스성 평가
Author
손지연
Alternative Author(s)
Son, Ji Yeon
Advisor(s)
이상경
Issue Date
2008-02
Publisher
한양대학교
Degree
Master
Abstract
RNAi란 siRNA가 RNA-induced silencing complx (RISC)와 결합해서 상보적인 유전자의 mRNA를 절개함으로써 그 유전자의 발현을 억제하는 현상이다. 이러한 siRNA의 특징을 이용하여 HIV 바이러스의 발현을 억제할 수 있는 최적의 항바이러스 벡터를 제조하였다. 특히 변형이 잘 일어나는 HIV의 특성을 고려하여 서열이 잘 보존된 최적화된 표적 유전자들을 선정하였고 각 변종에 관한 sequencing 결과, 숙주방어 회피를 돕는데 결정적인 역할을 하는 Vif (viral infectivity factor)와 HIV의 전 유전자 발현을 촉진하는 전사인자인 Tat (transcriptional activator)이 clade의 제한을 뛰어넘을 수 있는 가장 잠재력 있는 target임을 확인 할 수 있었다. 또한 다양한 virus mutant의 유전자 발현을 보다 효과적으로 억제하기 위해 두 개의 target을 동시에 발현할 수 있는 siRNA (bispecific miRNA)를 제조하여 감염된 세포에 발현시킴으로써 바이러스에 대한 완전한 protection이 가능함을 확인할 수 있었다. 특히, mRNA 절개에 가장 중요한 위치인 9, 10, 11 번 자리의 변형은 siRNA의 억제 효과를 두 배 이상 감소시키므로 이를 고려하여 바이러스의 자연 변종을 조사하여 인위적으로 변형된 서열을 luciferase reporter plasmid에 넣어 변종에 따른 siRNA의 억제 효과를 실험하였다. 그 결과, 선정된 두 siRNA 모두 중요 위치의 변형이 적을 뿐만 아니라 G:U wobbling base pairing에 의해 효과적인 바이러스 억제 효과를 보였다. 이러한 결과는 바이러스 치료의 가장 핵심적인 요인인 바이러스 변종에 대한 효율적인 치료 방안을 제시한다.; It has been shown that lentiviral delivery of monospecific short hairpin RNAs (shRNA) can stably suppress HIV-1 infection in primary T cells. However, given the requirement for sequence homology for RNAi-mediated gene silencing, viral heterogeneity is a major hurdle for its therapeutic use against HIV-1 since even a single nucleotide substitution, especially in the central target region (regions 8-12) can completely abolish silencing effect. Therefore, 5 simultaneously targeting multiple highly conserved viral sequences is thought to be the efficient way of safeguarding against viral propensity for escape from RNAi. Hence it becomes important to choose conserved RNAi targets where the possibility of occurrence of natural HIV variants having nucleotide changes in the central target regions is minimal. Keeping this criterion in mind, we have chosen 2 highly conserved 19 nucleotide target sequences in the vif and tat since computational analysis of sequences of 875 naturally occurring HIV variants in the Los Alamos Database revealed minimal sequence variations present at positions 9, 10 and 11 which could abolish silencing effects. Then we tested effects on silencing efficacy using artificial targets that reflect the natural variants occurring in HIV isolates corresponding to these target sequences. In addition, we also analyzed whether combinatorial targeting of vif and tat would prevent the generation of viral escape mutants. For this, we coexpressed these sequences as precursor microRNA (miRNA) stem-loop structures using a vector expressing pol II driven miR-155 miRNA backbone that allows efficient polycistronic expression of multiple miRNAs. We have confirmed siRNA expression by northern blots and successful inhibition of HIV by cotransfecting 293T cells 6 with NL4-3, a molecular clone of HIV and the miR construts. To test positional effects of mutations in target gene on inhibition efficacy, we generated artificial mRNA targets expressing the target sequences in fusion with luciferase mRNA as an indicator. The mRNA targets were based on sequences from naturally occurring HIV variants reported in Los Alamos Database. We found that the naturally occurring variants with nucleotide changes at the 9^(th) position, guanine to tyrosine (G9T) in tat or adenine (G9A) in vif are no longer responsive to silencing but the variant of vif at 11th position, adenine to guanine (A11G) was still amenable to inhibition, possibly due to wobble base pairing of the variant residue with the siRNA strand. In consistence with our hypothesis, replacing guanine with a cytosine (A11C) to remove wobble binding in target gene was resistant to silencing activity. Interestingly, there is no mutation at 10^(th) position in both target sequence. This might be indicatde that sequence alteration in this region might affect to viral fitness as well as 9^(th) and 11th since only a few variants are present in natural variants. However, bispecific siRNAs, miTat-Vif inhibited target constructs with a single variant at position 9. These results demonstrate that well designed bispecific miRNAs simultaneously targeting both tat and vif 7 regions could successfully inhibit quasi species of HIV and prevent generation of siRNA escaping variants because there is no naturally occurring variant virus has changes in sequence at position 9 in both tat and vif.
URI
https://repository.hanyang.ac.kr/handle/20.500.11754/147484http://hanyang.dcollection.net/common/orgView/200000408038
Appears in Collections:
GRADUATE SCHOOL[S](대학원) > BIOENGINEERING(생명공학과) > Theses (Master)
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