Gene therapy through bacteria
delivered RNA interference
Shuanglin Xiang
Key Laboratory of Protein Chemistry and Developmental Biology
of State Education Ministry of China,
College of Life Science, Hunan Normal University
The Nobel Prize in Physiology
or Medicine 2006
Andrew Z. Fire
Craig C. Mello
A natural existing mechanism
of sequence-specific posttranscriptional gene silencing
– Double-stranded RNA enters a target
cell, it is cleaved into short (~21nt)
fragments by DICER
– RISC (RNA-induced silencing
complex) separates these short
interfering RNAs duplex (siRNA), a
single strand RNA remains bound to
the complex
– RISC-associated single strand RNA
binds to homologous sequences in
mRNA within the cytoplasm
– This binding triggers the cleavage of
the target mRNA in the center of the
matched sequence
– Cleaved mRNA is quickly degraded
by cellular RNAses and can not be
translated into protein
Significance of the discovery of RNAi
1. RNAi protects against viral infections
2. RNAi secures genome stability by keeping mobile elements silent
3. RNAi-like mechanisms repress protein synthesis and regulate the
development of organisms
4. RNAi-like mechanisms keep chromatin condensed and suppress
transcription
5. RNAi offers a new experimental tool to repress genes specifically
6. RNAi might be a useful approach in future gene therapy
Top 10 Cancers in China
Male
Female
Lung cancer
Gastric cancer
Liver cancer
Colorectal cancer
Esophageal cancer
Pancreatic cancer
Bladder cancer
Brain cancer
Lymphoma
Nasopharyngeal cancer
Lung cancer
Breast cancer
Gastric cancer
Colorectal cancer
Liver cancer
Esophageal cancer
Ovarian cancer
Cervical cancer
Pancreatic cancer
Brain cancer
Delivery is a major problem
for RNAi in vivo application
(By Dr. Christopher P. Miller)
Disease – FAP (Familial Adenomatous Polyposis)
FAP is an autosomal, dominantly inherited disease that affects about 1 in 7000 individuals. Patients with FAP typically
develop hundreds to thousands of colorectal tumors (called adenomas or adenomatous polyps) during their second and third
decades of life. Although these benign tumors are not individually life-threatening, their large numbers virtually guarantee
that some will progress to invasive lesions (called cancer or carcinomas).
APC Min/+ mice
Cell 87: 159–170, 1996 and Science 275: 1787-1789, 1997
Bacteria delivered RNA interference
Cell Cycle 5:18, 2103
Bacteria deliver RNAi into C. elegans
( Original Idea, Fire A et al, 1998 )
a, General scheme for dsRNA production. Segments were cloned between flanking copies of the
bacteriophage T7 promoter into a bacterial plasmid vector. A bacterial strain (BL21/DE3) expressing the
T7 polymerase gene from an inducible (Lac) promoter was used as a host. As an alternative strategy, a
single copy of the T7 promoter were used to drive expression of an inverted duplication for a segment of
the target gene (gfp). A nuclease-resistant dsRNA was detected in lysates of these bacteria. b, A GFP
expressing C. elegans strain (PD4251) fed on a naive bacterial host. Animals show high GFP fluorescence in
body muscles. c, PD4251 animals reared on bacteria expressing dsRNA corresponding to the gfp coding
region. Under the conditions of this experiment, 12% of these animals show a dramatic decrease in GFP.
Bacteria
Cell
Transkingdom RNAi
Xiang, 2006
Construct for Transkingdom RNA Interference
TRIP carrying E. coli expresses shRNA and invades cells
“TROJAN HORSE” strategy
1.
2.
Interfering RNA can be manufactured by
bacteria (bacterial promoter)
Nonpathogenic bacteria actively invade target
cells deliver RNA interference
Bacterial steps involved:
•
•
•
•
Cell entry
Bacterial lysis
Bacterial RNAi liberation
Gene silencing through RISC
Interfering shRNA-expressing E. coli elicit potent RNAi
Induction of specific silencing in human colon cancer cells (SW480) by E.
coli expressing specific shRNA
Other factors: growth conditions
Effects of various concentrations of glucose on production of hemolysin
Influence of growth conditions on production of LLO
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1993, p. 3495-3497
pH affects the yield and activity of LLO
SCIENCE VOL 290 3 NOVEMBER 2000
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1993, p. 3495-3497
Temperature and pH affects the yield of Inv
Molecular Microbiology (2000) 35(3), 677-685
Oral administration or intravenous administration
of E. coli/TRIP-cat in animal experiments
I
b-catenin
II
shGFP
b-catenin
shCAT
PCNA
TK-RNAi targeted mouse b-catenin oncogene prevents growth and
malignant transformation of intestinal polyps in APC/min mice
b-Catenin
b-Actin
Control
shGFP
sh-mCat
“跨界基因沉默”技术的科学意义
1、
2、
3、
4、
在细菌中合成的小分子干扰RNA无须修饰和纯化，就可诱导哺
乳动物细胞特定基因的沉默；
将在遗传操作上简便易行、耗费低廉的细菌作为小分子干扰
RNA传递的媒介；
发现了益生菌在RNA干扰技术应用上的价值；
暗示了RNA干扰在不同物种间可能是相通的，细菌的存在对于
哺乳动物来说，不仅是一种共生的关系。
将开展的工作:
应用“跨界基因沉默”技术研制新型艾滋病疫苗;
人巨细胞病毒(HCMV)感染的治疗;
人乳头状瘤病毒(HPV)感染的治疗;
胞内菌清除机制的研究;
原核生物中RNA干扰现象的研究。
艾滋病毒感染的预防和治疗
I
b-catenin
Recombinant Escherichia coli Expressing Invasin Targets
the Peyer’s Patches
Selective invasion of PeyerTs patch tissue by invasive E. coli expressing GFP. C57/B6J mouse small intestines were
maintained in an organ bath and perfused with Krebs–Henseleit solution, E. coli BM2710 Prok.GFP (noninvasive, A, B, C)
or E. coli inv-hly/Prok.GFP (invasive, D, E, F). PeyerTs patches and segments of small intestinal tissue were excised, snap
frozen, and sectioned at a thickness of 5 Am. Paraformaldehyde-fixed sections were stained with DAPI (A, D) or with
hematoxylin and eosin (C, F). GFP (expressed by the E. coli) was visualized by fluorescence microscopy (B, E).
MOLECULAR THERAPY Vol. 14, No. 2, 2006
http://www.esnips.com/doc/246bbe09-2a92-4ff2-b9695e7d387d2830/Small-RNAs-Analysis-and-Regulatory-Functions
Contents
1.
Small Regulatory RNAs in Bacteria
2.
Small Nucleolar RNAs: Identification, Structure, and Function
3.
A Computational Approach to Search for Non-Coding RNAs in
Large Genomic Data
4.
Experimental Strategies for the Identification and Validation
of Target RNAs that Are Regulated by miRNAs
5.
Protein Interactions with Double-Stranded RNA in Eukaryotic Cells
6.
Transitive and Systemic RNA Silencing: Both Involving an RNA
Amplification Mechanism?
7.
RNA Interference and Antisense Mediated Gene Silencing
8.
Epigenetic Silencing of Transposons in the Green Alga
Chlamydomonas reinhardtii
9.
RNA-Dependent Gene Silencing and Epigenetics in Animals
10. Potentials of a Ribozyme-Based Gene Discovery System
谢 谢!