Interference and Inhibition:RNAi Mechanisms and Therapeutic Prospects

1
Interference and InhibitionRNAi Mechanisms and
Therapeutic Prospects
John J. Rossi-Division of Molecular
Biology Graduate School of Biological
Sciences-Beckman Research Institute of the City
of Hope,Duarte, California jrossi_at_bricoh.edu
2
RNA Interference

• First described in Plants (1992)as
  post-transcriptional gene silencing or
  co-suppression and later mechanism elucidated in
  C. elegans (1998).
• Subsequently shown to be active in mammalian
  cells (2001)
• Part of innate immune system
• Protection against double-stranded RNA viruses
• Protection against retrotransposon movement
• MicroRNAs regulate gene expression-800 to 1000
  miRNAs in humans, regulate expression of over 1/3
  of the genome!
• Chromatin silencing

1Fire A, et al. Nature. 1998391806-11.2Harborth
J, et al. J Cell Sci. 20011144557-65.
3
Post-transcriptional Cleavage of mRNA
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Processing Steps
miRNA microRNA dsRNA double-stranded RNA
siRNA short interfering RNA RISC RNA-induced
silencing complex ORF open reading frame
He L, Hannon GJ. Nat Rev Genet. 20045522-31.
5
Ago 2 binding of siRNA and passenger strand
cleavage.
PACT
Mammalian Dicer Co-factors areTAR RNA binding
protein and PACT-Chendrimada et al. 2005 Haase
et al. 2005) Lee, et al EMBO J. 2006.
6
Binding of miRNA or siRNA in Piwi domain of
Argonautes
A. fulgidus Piwi protein
Parker et al., Nature 2005
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How Is Antisense Strand Selected?
5'GCCNNNNNNNNNNNNNNNUAANN3'

3'NNCGGNNNNNNNNNNNNNNNAUU5'
Relative thermodynamic stability of 5' end
determines which strand is selectedweaker
pairing favors selection
Schwarz DS, et al. Cell. 2003115199-208.Khvorov
a A, et al. Cell. 2003115209-16.
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Cleavage Takes Place at Center of siRNA-Target
Duplex
5'
Cleavage of target directed by middle of siRNA
measured 10 nucleotides from 5' end of siRNA
AAAn
NAAGCCUGUGCCUCUUCAGCUACC
UUCGGACACGGAGAAGUCGAUGp5'
11,10
Mismatches at site of cleavage inactivate siRNA,
others are tolerated to various extents
Hutvagner G, Zamore PD. Science.
20022972056-60.
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Applying RNAi for the treatment of HIV infection

• Several early reports demonstrating that HIV was
  a highly susceptible target for RNAi
• Can use RNAi in a gene therapy setting by
  expressing short hairpin triggers.

Lee N. et al. 2002, Nat. Biotechnol. (2002) Li M.
et al. 2005, Mol Ther (2005) Unwalla et al. J.
Virol. (2006)
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Triggering RNAi in Mammalian Cells
shRNA short hairpin RNA siRNA short
interfering RNA RISC RNA-induced silencing
complex
Scherer LJ, Rossi JJ. Nat Biotechnol.
2003211457-65.
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Choosing the best vector for therapeutic gene
delivery
Lentiviral vector
Rev
Gag Pol
VSV-G
Packeging cell
LTR self-inactivating Lack of viral
genes InfectT-cells and non-dividing
hematopoietic progenitor cells
Target cell
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Lentiviral vector construct stably expressing
shRNAs
U6 S / U6 AS siRNA
LTR
LTR
EGFP
CMV
RRE/PPT
U6 promoter
sense
loop
antisense
ter
siRNAs
5-GCGGAGACAGCGACGAAGAGCTTTGTGTAGGCTCTTCGTCGCTGTCT
CCGCTTTTTT
5-------GCGGAGACAGCGACGAAGAGCUU
UUCGCCUCUGUCGCUGCUUCUCG-------5
U
U
U
G
5-GCGGAGACAGCGACGAAGAGC 3-UUCGCCUCUGUCGCUGCU
UCUCG
S/AS(I)
U
G
U
A
G
siRNA
13
tat/rev - Site 1 siRNA
rev Site 2
Rev
Tat
Gag
LTR
LTR
Vif
Env
Nef
Pol
Vpr
Vpu
14
(No Transcript)
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Reverse Transcriptase units
siRNA synergizes with AZT
16
Emergence of a viral mutants resistant to shRNA
Under certain in vitro culture conditions, RNAi
escape mutants emerge when only a single shRNA is
being utilized.
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Wild type-5 GCGGAGACAGCGACGAAGAGC
MTB 5 GCGGAAACGGCGACGAAGAGC
MTC 5
GCGGAGAAAGCGACGAAGACC MTD
5GCGGAACCAGCGACGAAGAGC
MTE 5 GUGGAGACAGCAACGAAGAGC
Some tat/rev variants isolated with shRNA
selective pressure
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G C G G A G A C A G C G A C G A
A G A G C T C A T C A G A A C
A G T C A GACTC
Mutant grown on shRNAtat/rev expressing cells,
strong selection for mutant.
Mutant grown on untransfected cells, wild type
comes back as predominant species.
5-GCGGAGACAGCGACGAAGAGC3
3-UUCGCCUCUGUCGCUGCUUCU5
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Combinatorial RNA based therapy for HIV

• Most efficacious drug therapy for HIV uses
  combination of two or three drugs targeting HIV
  RT and protease. Combinations prolong and
  sometimes prevent resistant viral variants.
• Can effective combinatorial gene therapy from a
  single vector be accomplished?

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Rationale for anti-HIVGene Therapy

• HIV infection is chronic and lifelong treatment
  required.
• Increasing number of patients with drug resistant
  virus.
• Toxicities of current chemotherapy agents (in
  Highly Active Anti-Retroviral Therapy cocktail).
• Difficult dosing regimens for patients
• Expense of HAART can exceed 20,000/yr
• Gene Therapy can replace or be used as an
  adjuvant for chemotherapy

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Isolated CD4 Cells
T cell-based gene therapy
Transduction of CD4 cells with genetic vectors
CD4 Cell Selection and Expansion
Relatively easy to obtain and genetically modify,
but does not capture all of viral reservoirs such
as monocytic and dendritic cells.
Infusion
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Stem cell-based gene therapy
Transduction targets
NK-Pre
NK cell
Pre-B
Plasma Cell
B cell
Lymphoid stem cell
T cell
Pre-T
Pluripotent Stem Cell
CFU-Blast
CFU-G
PMNL
CFU-GM
CFU-M
Monocyte
CFU-GEMM
BFU-Meg
Megakaryocyte
Hematopoiesis
Retic
RBC
BFU-E
CFU-E
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Combinatorial therapeutic RNAs

• Nucleolar localizing TAR decoy
• (Michienzi et al. 2002, 2006)
• Chimeric VA1CCR5 ribozyme
• (Cagnon et al, 1997 Li et al., 2003)
• Anti tat/rev shRNA

Each of these RNAs inhibits HIV-1 by a different
mechanism Therefore it may be advantageous to
combine these in a therapeutic setting
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Nucleolar localizing TAR decoy inhibition of HIV
U16TAR
U3
A
U6 prom
U16TAR
6T
U16TAR/
U3
DAPI
14000
U16Tar 1
U16TAR
U16Tar 2
12000
1
2
3
4
5
6
7
8
U16Tar 3
10000
U16Tar 4
8000
HIV RT Unit/ml
U16Tar 5
6000
U16Tar 6
U16TAR
U16Tar 7
4000
U16Tar 8
U16
2000
U16Rz wt
U16Rz
0
tRNA
lys
8
14
21
1
2
3
4
5
6
7
8
9
Days post-infection
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Therapeutic genes (cont.)
Anti-CCR5 Ribozyme
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Down Regulation of CCR5 Transcripts in Primary
monocytes by Anti-CCR5 Ribozyme
A
shI-TAR-RZ
shI-RZ
Control
shI
CCR5
GAPDH
B
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(No Transcript)
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pHIV-7-GFP
flap
CMV
EGFP
R
U3
U5
CMV
RRE
R
U5
?
WPRE
U6
shI
pHIV-shI-GFP
U6
TAR
U6
shI
pHIV-shI-TAR-GFP
VA1
RZ
U6
shI
pHIV-shI-CCR5RZ-GFP
VA1
RZ
U6
TAR
U6
shI
pHIV-shI-TAR-CCR5RZ-GFP
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Method of Experimentation
CD34 cord blood or peripheral stem cells
Transduction with vector
7 days
Sorting by FACS
GM-CSF and M-CSF 8 days
FACS Characterization Macrophage Function HIV
challenge
Mature monocyte/macrophages
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Expression of Therapeutic Genes in Target Cells
CD34
CEM
shI-TAR-CCR5RZ
shI-TAR-CCR5RZ
Unrelated shRNA
Non-transduction
shI-CCR5RZ
GFP only
GFP only
shI-TAR
shI
shI
- CCR5RZ
- TAR
- U6
- shI
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Triple construct transduced hematopoietic stem
cells differentiate normally into
monocyte/macrophage and dendritic cells
80
70
60
Control
50
GFP
Percent cells
shS1
40
Triple
30
20
10
0
CD11c BDCA4
CD14
CD11c
BDCD4
CD14 CD11c
CD14 BDCA4
CD11c-
BDCA4
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Potent inhibition of HIV-1 replication
Triple combination provides better protection
than individual shRNAs in transduced CD34
derived monocytes/macrophages
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HIV-1 challenge of triple transgenic thymocytes
-
60 days
Purify T cells
HIV-1 challenge
Triple vector transduced CD34 cells
differentiate into thymocytes in vivo in SCID-hu
mice. T-lymphocytes are resistant to viral
challenge.
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Summary of Preclinical Testing

• Long term protection against HIV replication in
  primary CD34 derived monocytes and macrophatesl
  (Li et al., Mol. Therapy 2005)
• Lack of immunogenicity and normal myeloid
  differentiation Robbins et al., Nature Biotech
  2006.
• Normal thymic T-cell development and resistance
  to T-tropic HIV (Anderson, et al Mol. Therapy
  2007).

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1996 established autologous BMT for AIDS
lymphoma
From A. Krishnan et al. Blood 2005 105874-8
Initial 5 patient study demonstrated safety and
feasibility of gene therapy in AIDS lymphoma
patients.Overall 85 survival rate for autologous
stem cell transplants in AIDS lymphoma patients.
Relapse rate without BMT exceeds 50. New stem
cell transduction protocols and new vectors
provide the impetus using this patient population
in new AIDS gene therapy trial.
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Clinical Protocols Schema
CD34 Stem Cell Enrichment
Fibronectin and growthfactors
HIV patient
Apheresis
PBMCs
CD4 T-cell enrichment, transduction, expansion
and reinfusion
Transduction
G-CSF RX
Preparation for autologous BMT
Pool unmodified and modified cells and infuse

Cell engraftment and survival
Patient Follow up
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Steps to the clinic

• Create final version of clinical vector by
  deletion of EGFP-develop real time PCR based
  assay for titering vector.
• Scale up production of clinical vector.
• Optimize CD34 transduction protocols.
• Analyze vector integration sites.
• Perform microarray analyses of CD34 triple
  construct transduced versus vector backbone
  transduced cells.

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Steps to the clinic for HSC trial

• Presentation to and meeting with NIH Recombinant
  DNA Advisory Committee Sept. 2005-on NIH-RAC web
  site
• File FDA application Jan. 2007
• Final approval by FDA May 30-2007
• 1st Patient enrolled June 2007-infusion of
  autologous stem cells mid July.

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Center for Biomedicine and Genetics
Beckman Research Institute at City of Hope
Clinical grade triple vector production is
complete, with enough vector to treat 6 BMT and 6
autologous T-cell patients
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Trial highlights

• 1st human clinical trial using lentiviral vector
  transduction of HSCs.
• 1st human trial with expressed RNA interference
  trigger (shRNA).
• 1st triple gene therapy combination trial for
  HIV/AIDS.

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Acknowledgments
Mingjie Li Marjorie Robbins, Haitang Li Daniela
Castanotto Nancy Lee Lisa Scherer Alessandro
Michienzi Laurence Cagnon
COH collaborators John ZaiaLarry Couture
Shirley Li, Priscilla Yam David HsuDavid
DiGuisto Ramesh Akkina (CSU)
NIH NIAID, NHBLI Benitec, Inc.