Name: evaluation of the efficacy and toxicity of rnas targeting hiv-1 production for use in gene or drug therapy
Uploaded: 2016-09-05T14:00:00.000+00:00
Duration: 12 min 3 s
Description: Watch this Scientific Journal Video about Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy at JoVE.com

여기에 제시된 작품은 건강 연구 (CIHR)의 캐나다 연구소에 의해 지원되었다 (DCB-120266, PPP-133377와 AG에 HBF-348967을 부여).

1. 세포와 형질 <ol><li> 둘 베코 변성 이글 배지 (DMEM)에서 배양 HEK 293T 세포를 10 % 소 태아 혈청 (FBS) 및 1 % 페니실린 / 스트렙토 마이신이 보충. 세포 배양 배지에서 2 × 105 세포 / ㎖ 현탁액을 준비한다. 의 각 웰에 세포 현탁액의 500, 100 및 1,000 μl를 추가, 24도, 96도, 12 웰 플레이트, 바이러스 생산, 세포 생존 및 면역 활성 ​​분석, 각각 (그림 2A)합니다. </li><li> 부드럽게 플레이트 소용돌이와 5 % CO 2와 37 ° C에서 / O N 그들을 품어. 50~70%의 포화 상태로 세포를 성장. </li><li> 형질 전환 계획에 따르면, 1.5 ml의 마이크로 튜브 (예, 그림 2B)에 테스트 RNA를 자신의 컨트롤의 희석을 준비합니다. <ol><li> 바이러스 생산 분석를 들어, HIV-1 발현 플라스미드의 10 NG / μL 희석을 준비하고 각 튜브에 10 μl를 추가합니다. 다음 5 μM 시험의 RNA 희석하여 음성 대조군 RN 준비A. 25 또는 100 nM의 최종 농도에 해당하는 튜브에 각 시험 RNA 및 음성 대조군 희석 2.5 또는 10 μl를 추가합니다. </li><li> C : 세포 생존력 분석을 위해 5 μM 시험의 RNA 희석하고 양성 대조군 RNA, 저 분자량 폴리 I의 10 ㎎ / ㎖의 희석을 준비한다. 각각 100 nm의 또는 200 μg의 / ml의 최종 농도에 해당하는 튜브에 테스트 RNA 또는 양성 대조군 RNA 희석 2 μl를 추가합니다. </li><li> 면역 활성 시험의 경우, 시험 RNA 또는 단계 1.3.2에서 제조 한 양성 대조군 RNA 희석 20 μl를 추가합니다. 각각 100 nm의 또는 200 μg의 / ml의 최종 농도에 해당하는 튜브에. 단계 1.3.1에 ​​대한 ng를 25, 100의 최종 금액을주고, 테스트 RNA 발현 플라스미드를 들어 시험의 RNA의 5 μM 희석 대신에 10 NG / μL 희석을 준비 :합니다. 및 (100)는 단계 1.3.2에 대한 ng를. 및 1.3.3. </li></ol></li><li> 바이러스 성 자극에 대한 각각의 형질 튜브에 DMEM 50, 25 또는 75 μl를 추가각각 uction, 세포 생존 및 면역 활성 분석. 바이러스 생산 분석법 다음 단계 전에 생물학적 안전 수준 3 (BSL3) 실험실 세포 형질 제조 튜브를 가져온다. </li><li> 트랜 튜브에 형질 전환 시약 순차적으로 2 μl를 추가하고 복합체 형성 할 수 있도록 15 ~ 20 분을 품어. 특정 형질 전환 시약 재료의 표를 참조하는 데 사용합니다. </li><li> 적가 세포 배양 플레이트의 상응하는 위치에 각각의 마이크로 튜브에서 전체 형질 전환 혼합물을 추가한다. 부드럽게 소용돌이와 5 % CO 2, 37 ℃에서 48 시간 동안 접시를 품어. </li><li> 세포 배양 판 (그림 2C)에서 HIV-1 생산 (섹션 2), 세포 생존 능력 (섹션 3) 및 면역 활성화 (섹션 4)를 측정한다. </li></ol> 2. 바이러스 생산 분석 <ol><li> 배양기에서 24 웰 세포 배양 플레이트를 제거하고 BSL3 세포 배양 플레이트의 내부 소용돌이 부드럽게후드. A는 HIV-1 생산을 정량화하는 데 사용됩니다 96 웰 평평한 바닥 플레이트에 잘 대응하는 각 우물에서 뜨는 150 μl를 전송합니다. 주 : 일반 바이러스 정량 분석은 효소 면역 분석법 (ELISA) (20), 역전사 중합 효소 연쇄 반응 (RT-PCR) (21)에 의한 바이러스 RNA를 정량하여 HIV-1 캡시드 단백질 (P24)의 발현을 측정하여 활성을 측정 포함 에이즈-1 RT 효소. 2.2 2.5은 HIV-1 RT 활동 13,15,16를 정량화하는 방법을 설명하는 단계를 반복합니다. </li><li> 전송 중단 바이러스 칵테일 15 (표 1) 25 μl를 함유하는 96 웰 플레이트에 웰 대응하는 상등액 5 μL. RT에서 5 분 혼합물을 품어하고 방사능 워크 스테이션에 접시를 전송합니다. 참고 : 2.2 단계. 방사능 워크 스테이션이 BSL3 실험실에서 사용할 수없는 경우에만 필요합니다. 플레이트로부터 제거 할 필요가없는 경우BSL3 실험실, 2.3 단계로 진행합니다. 방사성 칵테일의 25 μL 대신 바이러스 / 방사성 붕괴 칵테일 (표 1) 50 μl를 추가합니다. </li><li> 방사성 칵테일 (15) (표 1)를 준비하고, 바이러스 뜨는 및 중단 칵테일의 각 웰에 25 μl를 추가합니다. 2 시간 동안 37 ° C에서 접시를 품어. </li><li> 스팟 5 유리 섬유 디 에틸 아미노 에틸 (DEAE) filtermat 용지에 해당하는 사각형 상 반응 혼합물의 μL 및 관광 명소가 10 분 동안 건조 할 수 있습니다. 두 개의 샘플들이 직접 서로 보더 없도록 다른 모든 사각형 반응 혼합물 스팟. 이 단계 2.6 분 (CPM) 당 수를 결정할 때 샘플 사이의 크로스 오버를 방지하는 데 도움이됩니다. </li><li> 종이 95 % 에탄올이 1 분간 세척 한 다음 염수 배 구연산 나트륨 (SSC) 버퍼 (표 1)을 5 분 동안 5 배 씻는다. 종이 건조 및 샘플 가방을 봉인 할 수 있습니다. </li><li> 클립 샘플 가방 공동카세트에 filtermat 용지를 ntaining 및 마이크로 플레이트 섬광 계수기에 카세트를 삽입합니다. 실험에 사용 [32 P] dTTP를의 일괄 제공 기준 날짜 (32) P에 대한 CPM을 읽고 카운터를 설정합니다. 플레이트지도를 이용하여 판독하고 카운터를 시작하는 샘플을 선택한다. </li><li> 모든 바이러스의 정량 방법, 인접 음성 대조군으로 각각 테스트 RNA에 대해 얻어진 값을 나누어 테스트 RNA를 각 복제에 대한 HIV-1 생산의 억제 백분율을 얻기 위해 (100)에 의해이 값을 곱한다. 다른 농도에서 다른 테스트 RNA를 비교 결과의 일례는도 3에 제공된다. </li></ol> 3. 세포 생존 분석 <ol><li> / ml의 MTT (3- [4.5 디메틸 -2- 티아 졸릴] -2,5- 디 하이드로 -2H- 테트라 졸륨 브로마이드) 둘 베코 인산염 완충 식염수에 희석 (인큐베이터에서 96 웰 플레이트를 제거하고 5 mg의 20 μL를 추가 각 웰에 DPBS). F 번호판을 품어또는 37 ℃에서 3 시간. </li><li> 세제 (1 % NP-40, 이소프로판올 4 mM의 염산)도 각으로 산성화 이소프로판올 150 μl를 추가하고 실온에서 2 시간 동안 접시를 품어. </li><li> 마이크로 플레이트 분광 광도계에서 570 nm에서 흡광도를 결정합니다. </li><li> 인접 형질 전환 제어에 의해 각각의 샘플에 대해 얻어진 값을 나누어 각각 양성 대조군 및 테스트 RNA에 대한 상대 MTT 대사를 계산한다. 다른 테스트의 RNA와 양성 대조군을 비교 한 결과의 일례는도 4에 제공된다. </li></ol> 4. 면역 활성화 분석 <ol><li> 인큐베이터에서 12 웰 플레이트를 제거하고 문화 매체를 대기음. 부드럽게 DPBS로 두 번 세포를 씻어 아니라 각 (프로테아제 및 포스 파타 아제 억제제, 표 1 포함) 차가운 용해 버퍼 (22)의 70 μl를 추가합니다. 얼음에 10 분 동안 접시를 품어. </li><li> 마이크로 튜브에 세포 용 해물을 전송하고 빠른 침지하여 그들을 동결액체 질소에서 튜브. 샘플은 해동 3의 총이 더 동결 해동 사이클을 반복 할 수 있습니다. </li><li> 세포 파편을 펠렛, 4 ° C, 15,700 XG에서 15 분 동안 해물을 원심 분리기. 새로운 마이크로 튜브에 뜨는을 전송하고 쿠마 블루 (브래드 포드) 메소드 (23, 24)를 사용하여 단백질 농도를 결정한다. </li><li> 10 % 변성 폴리 아크릴 아미드 겔에서 각 샘플로부터의 단백질 75 μg의 해결 이전 25,26 바와 같은 니트로 셀룰로오스 막에 옮긴다. </li><li> 막에 전기 및 전송 후, 증류수에 세척 한 다음 1 분 동안 폰소 s (표 1)에서 막을 배양하여 단백질 밴드를 보여준다. 80, 55 kDa의에서 막을 잘라 가이드로 밴드와 단백질 사다리를 사용합니다. 주의 : 단계 4.4 샘플 16 × 18cm 34 kDa의 아래로 2 겔을 실행할 수 있으며,이 지정된 위치에 막을 잘라 통해 절단되지 쉽다 있도록관심의 밴드. 대안 적으로, 몇몇 겔 막을 절단 피하기 위해 동일한 샘플을 실행할 수있다. </li><li> 0.05 % 트윈 20 (TBST, 표 1)를 포함하는 TBS 버퍼로 폰소 s 염색을 세척 할 것. 완전히 막 다루 5 % 무 지방 우유로 TBST를 추가합니다. 1 시간 동안 교반하면서 RT에서 막을 인큐베이션. </li><li> 막 부화 O / N ADAR1 (110 및 150 kDa의) 포스 PKR (62 kDa 이상) 및 포스 IRF3 (47 kDa의)에 대하여 1000 년 1로 희석하고 3 % 소 혈청 알부민 TBST (BSA)과 항체에있어서, 각각 멤브레인의, 상단 중앙과 하단 부분에 대해. </li><li> 멤브레인은 TBST 5 분 동안 5 배 1 시간 동안 (5000 년 1로 희석) 5 % 무 지방 우유와 퍼 옥시 다제 표지 염소 항 - 토끼 보조 항체와 TBST에 품어 씻으십시오. </li><li> TBST 5 분 동안 막 배를 세척하고 제조업체의 지침에 따라 영화에 밴드를 시각화하는 전기 화학 발광 (ECL) 솔루션을 적용 할 수 있습니다. </li><li> 필름의 단백질 밴드를 가시화 한 결과, 항체 박리 용액으로 10 분 동안 멤브레인의 중간 및 하단 부분을 세척한다. 및 IRF3 (500 1) 총 PKR에 대해 3 % BSA와 TBST에서 세포막 O / N 및 항체를 품어 (1,000에서 1에서) 각각 중앙과 하단 부분에 대해. 반복 4.8 단계를 반복합니다. 4.9. PKR 막 (중간)에 대한 항 - 토끼 이차 항체 대신에 퍼 옥시 다제 표지 된 염소 항 - 마우스를 사용. </li><li> TBST 5 분 동안 막 5 배의 바닥 부분을 세척하고 (5000 년 1로 희석) 굴지에 대한 항체와 1 시간 동안 3 % BSA와 TBST에서 막을 품어. 반복 4.8 단계를 반복합니다. 4.9. 항 - 토끼 이차 항체 대신에 퍼 옥시 다제 표지 된 염소 항 - 마우스를 사용. 다른 테스트의 RNA와 양성 대조군을 비교 한 결과의 일례는도 5에 제공된다. 사용되는 특정 항체 물질의 표를 참조. </li></ol>

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The authors have nothing to disclose.

기재된 HIV-1 생산 분석법 HEK293T 셀들 (도 2)를 이용하여 수행하고, 효과적인 리보 자임 (13) shRNA를 10,29, siRNA를 30 및 U1i RNA 11,31 표적 부위를 HIV-1 RNA를 선별하는 데 사용되는 분석 비슷 하였다. HIV-1 생성을 정량화하기 위해 다양한 방법을 사용하여, 대부분의 연구는 후보의 RNA와 HIV-1 발현 플라스미드의 공동 형질 감염 후 바이러스 생산을 48 시간으로 측정...

현재 HIV-1 치료의 한계는 만성적 질환의 진행을 방지하기 위해 투여되어야한다는 것이다. HIV-1에 강한 T 림프구 또는 조혈 줄기 세포 이식, 약물 치료 -1,2-의 부재 HIV-1 복제의 장기간 제어를 제공하는 가능성을 가지며 또한 HIV-1 치료를 달성하기위한 효과적인 방법이 될 수있다 3. HIV-1 복제에 대해 내성 세포를 렌더링하기위한 한 가지 방법은자가 이식 동안 4 감염된 개인의 세포에 항 HIV-1 RNA를 또는 펩타이드를 코딩하는 하나 이상의 유전자를 삽입하는 것이다. 여러 후보 항 HIV-1 유전자는 단일 유전자에 HIV-1의 저항 현상을 방지하기 위해, 두 개 또는 세 개의 5 (6)의 조합에 일부 진입하여 임상 시험을 고안 하였다. 항 HIV-1 RNA를 인해 면역 반응을 유도하는 그들의 낮은 전위로하고 있기 때문에 결합 유전자 치료에 대한 상위 후보자들이다매우 짧은 유전자 서열로부터 전사된다. 일부 항 HIV-1 RNA를이 바이러스 항목 및 통합을 목표로 설계되었습니다. 그러나, 대부분의 항 HIV-1 RNA를는 바이러스 라이프 사이클 후 통합 단계 (도 1)을 대상. 후 통합 억제제는 리보 자임 (7), shRNA를 8 U1i RNA를 9로는 HIV-1 조절 단백질 문신 또는 계 1, 및 안티센스 기반의 RNA를 대상으로 HIV-1 RNA에 다른 사이트를 대상으로, 미끼 RNA를 포함. 항 HIV-1 RNA를의 효능을 비교하기 위해 사용 된 방법은 유전자 후보 RNA를 코딩하는 바이러스 성 일시적 후보의 RNA를 발현하는 플라스미드로 형질 감염된 세포에서 생산 및 HIV-1 발현 플라스미드를 측정 형질 세포에서 바이러스 복제를 모니터링 포함 10 -13. 우리는 이전에 새로운 리보 자임 대상 사이트 13 ~ 15에 대한 HIV-1 RNA를 선별하는 HIV-1 생산 분석을 사용했다. 이러한 방법은 RNA 인 이후의 포맷을 최적화하기 위해 정제 된간섭은 분자의 shRNA로 플라스미드 DNA로부터 발현 또는 합성 siRNA를 16로 전달. 상기 분석은 인간 배아 신장 (HEK) 293T 세포로부터 성숙 바이러스의 생산을 측정하고 (도 1) HIV-1 복제 사이클 후 통합 단계를 타겟팅 억제제의 효과를 비교하기 위해 사용될 수있다. 사전 통합 단계를 타겟팅 억제제를 들면, TZM-BL 세포 감염 분석 (17)와 같은 다른 분석은 항 바이러스 효능을 평가하기 위해 필요하다. 병원에서 항 HIV-1 RNA를의 전달을위한 주요 안전 문제는 인간의 RNA 또는 단백질과 선천성 면역 센서의 작동에 잠재적 인 오프 대상 효과를 포함한다. 항 HIV-1 siRNA의 독성을 평가하기 위해 다른 세포주 16 세포 생존력 분석을 이용했다. 우리는 또한 이중 가닥 RNA 면역 센서의 활성화를 측정 RNA 활성화 단백질 키나제 R (PKR) 및 수용체 3 (TLR3) 등 수신자뿐 아니라 전인터페론의 Pression의 유전자, ADAR1의 P150을 자극. 이러한 분석은 항 HIV-1 RNA를 효능은 세포 생존 또는 면역 센서의 활성화에 대한 간접적 인 효과에 기인 아니라는 것을 확인하는데 사용될 수있다. 또한 발전에서 잠재적 인 독성과 후보 RNA를 제외 유용하다. 다음 프로토콜에서, 절차는 새로운 치료의 RNA를 확인하고, 기존의 형식을 설명하는 최적화. 방법은 HIV-1 복제의 심사 RNA 기반 후 통합 억제제에 유용과 같은 바이러스 성 RNA 18 CRISPR의 레브 매개 수출을 대상으로 작은 분자와 같은 다른 후 통합 억제제를 화면에 적용 할 수 / 설계 카스 시스템은 통합 대상으로 HIV-1 DNA 19.

<table><tbody><tr><td>DMEM HyClone</td><td>GE Healthcare</td><td>SH30243.01</td><td></td></tr><tr><td>FBS HyClone</td><td>GE Healthcare</td><td>SH30396.03</td><td></td></tr><tr><td>Penicillin/Streptomycin Gibco</td><td>Thermo Fisher</td><td>15140-122</td><td></td></tr><tr><td>Cell culture plates, 96 well, 24 well, 6 well.</td><td>Corning</td><td>353075, 353047, 353043</td><td></td></tr><tr><td>Micro tubes Axygen</td><td>Corning</td><td>311-08-051</td><td></td></tr><tr><td>Low molecular weight Poly I:C</td><td>InvivoGen</td><td>3182-29-6</td><td></td></tr><tr><td>DharmaFECT-1</td><td>Dharmacon</td><td>T-2001-01</td><td>transfection reagent for synthetic RNAs</td></tr><tr><td>TransIT-LT1</td><td>Mirus</td><td>MIR 2300</td><td>transfection reagent for RNA expression plasmids</td></tr><tr><td>Nonidet P40 (NP-40)</td><td>USB</td><td>19628</td><td></td></tr><tr><td>[&lt;sup&gt;32&lt;/sup&gt;P]dTTP</td><td>Perkin Elmer</td><td>BLU505H</td><td></td></tr><tr><td>poly(A) RNA template&amp;nbsp;</td><td>Sigma-Aldrich</td><td>10108626001</td><td></td></tr><tr><td>oligo(dT)&lt;sub&gt;12-18&lt;/sub&gt; DNA primer</td><td>Thermo Fisher</td><td>18418-012</td><td></td></tr><tr><td>DEAE filtermat paper&amp;nbsp;</td><td>Perkin Elmer</td><td>1450-522</td><td></td></tr><tr><td>Microplate scintillation counter</td><td>Perkin Elmer</td><td>1450-024</td><td></td></tr><tr><td>MTT</td><td>Sigma-Aldrich</td><td>M-2128</td><td></td></tr><tr><td>DPBS HyClone</td><td>GE Healthcare</td><td>SH30028.02</td><td></td></tr><tr><td>Microplate spectrophotometer</td><td>Bio-rad</td><td>1706930</td><td></td></tr><tr><td>Lysis buffer tablets</td><td>Roche</td><td>4693159001, 4906837001</td><td>protease and phosphatase inhibitors</td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5415R</td><td></td></tr><tr><td>Bradford reagent</td><td>Bio-rad</td><td>500-0006</td><td></td></tr><tr><td>Gel running chamber</td><td>Hoefer</td><td>SE600</td><td></td></tr><tr><td>Semi-dry transfer cell</td><td>Bio-rad</td><td>1703940</td><td></td></tr><tr><td>Protein ladder EZ-Run</td><td>Thermo Fisher</td><td>BP3603-500</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>Bio-rad</td><td>162-0094</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A9647-1006</td><td></td></tr><tr><td>Antibody stripping solution</td><td>Millipore</td><td>2504</td><td></td></tr><tr><td>ECL - Pierce</td><td>Thermo Fisher&amp;nbsp;</td><td>PI32106</td><td></td></tr><tr><td>ADAR1 antibody</td><td></td><td></td><td>from Dr. B.L. Bass</td></tr><tr><td>phospho-T446-PKR antibody</td><td>Abcam</td><td>ab32036</td><td></td></tr><tr><td>phospho-S396-IRF3 antibody</td><td>Cell Signaling</td><td>4947</td><td></td></tr><tr><td>PKR antibody</td><td></td><td></td><td>from Dr. A. Hovanessian</td></tr><tr><td>IRF3 antibody</td><td>Cell Signaling</td><td>11904</td><td></td></tr><tr><td>Actin antibody</td><td>Millipore</td><td>MAB1501</td><td></td></tr><tr><td>Peroxidase-labeled goat anti-rabbit</td><td>KPL</td><td>474-1506</td><td></td></tr><tr><td>Peroxidase-labeled goat anti-mouse</td><td>KPL</td><td>474-1806</td><td></td></tr><tr><td>Ponceau S&amp;nbsp;</td><td>Sigma-Aldrich</td><td>6226-79-5</td><td></td></tr></tbody></table>

evaluation of the efficacy and toxicity of rnas targeting hiv-1 production for use in gene or drug therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

절차의 일반적인 개략도 세 시험 RNA를도 2b에 제공되는 제어 RNA위한 예시 형질 계획도 2에 도시되어있다. 바이러스 생산 및 세포 생존 분석을 위해 각 테스트 구조에 대한 판독은 음성 대조군으로 정규화된다. 각 시험 RNA가 인접 대조군에 정상화되도록 복제가 세트로 형질 감염된다. 이것은 예를 들어, 음성 대조군 모두 제 형질, 경우 발생할 수 착...

Watch this Scientific Journal Video about Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy at JoVE.com

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

유전자 또는 약물 치료에 사용하기 위해 HIV-1 생산을 표적 RNA를의 효능 및 독성 평가

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

evaluation-efficacy-toxicity-rnas-targeting-hiv-1-production-for-use

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

7.8K 조회수.  Lady Davis Institute for Medical Research. Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones. 

비디오: Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

RNAi 게놈 넓은 검사 Oncolytic 바이러스 치료 변조 호스트 요소를 식별 하

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

연구

암 연구학

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

siRNA 전달을위한 셀 타입의 특정 안티 HIV gp120 aptamers의 개발

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

면역학 및 감염병학

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

암 관련 유전자 기능을 연구하기 DNA를 벡터 기반의 RNA 간섭

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

의학

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA 바이러스의 넓은 스펙트럼 화학 억제를위한 높은 처리량 검사

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV 역전사 및 인테그라 제 억제제의 빠른 검사

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

RNAi Effectors의 세균성 배송 : Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

생물학

MicroRNA-based Regulation of Picornavirus Tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

피 코르 나 바이러스과의 바이러스 친 화성의 마이크로 RNA-기반 규제

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

대기 시간 반전 HIV 전사 및 접합에 에이전트의 체 외 평가에서 높은 처리량

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

형광에 의한 바이러스성 RNA의 탐지<em&gt; 현장에서</em&gt; 하이브리드화 (물고기)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. Although combinational antiretroviral therapy (cART) has been successful in suppressing viral replication, it cannot completely eradicate the reservoir from HIV-infected individuals. A safe and effective cure strategy for HIV infection will require multipronged methods, and therefore the advancements of animal models for HIV-1 infection are pivotal for the development of HIV cure research. Humanized mice recapitulate key features of HIV-1 infection. The humanized mouse model can be infected by HIV-1 and viral replication can be controlled with cART regimens. Moreover, cART interruption results in a prompt viral rebound in humanized mice. However, administration of cART to the animal can be ineffective, difficult, or toxic, and many clinically relevant cART regimens are unable to be optimally utilized. Along with being potentially unsafe for researchers, administration of cART by a commonly used intensive daily injection procedure induces stress by physical restraint of the animal. The novel oral cART method to treat HIV-1 infected humanized mice described in this article resulted in suppression of viremia below the detection level, increased rate of CD4+ restoration, and improved overall health in HIV-1 infected humanized mice.

This protocol describes a novel method to deliver oral combinational antiretroviral drugs that successfully suppress HIV-1 RNA replication in humanized mice.

HIV-1에 감염된 인간화 마우스에서 경구 조합 항레트로바이러스 치료

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. ...