Download Genome Editing-Based HIV Therapies

Genome Editing-Based HIV
Therapies
 Rehana Kousar
 PhD Scholar
 06-arid-746
 Department of Botany
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Trends in Biotechnology
Review
Genome Editing-Based HIV Therapies
Wang-Gang Gu
15 January 2015
• Genome editing (GE)-based HIV therapy is achieved by
modification of infection-related genes to produce HIVresistant cells followed by reinfusion of the modified cells
into patients. The ultimate goal is to achieve a functional
or actual cure for HIV infection. Despite multiple
potential targets for GE-based HIV therapies,CCR5 is the
most feasible owing to the naturally existing CCR5 δ32
genotype which confers resistance to HIV. A recent
clinical trial of infusion of modified autologous CD4+ T
cells proved safety and efficacy within the limits of the
studies. However, long-term evaluation of the safety and
efficacy is required before GE-based HIV therapy is ready
for clinical implementation.
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Contents
 HIV
 HIV Infection
 Clinical treatment of AIDS
 GE-based Therapies
 Advances in GE-based Therapies
 Targets of GE-based Therapies
 Concerns in GE-based Therapies
 Conclusions
 Future perspective
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HIV
 The
Human Immunodeficiency Virus
a lentivirus that causes AIDS i-e.,
immunodeficiency syndrome
(HIV) is
acquired
 A serious infectious disease characterized by the
systemic collapse of the immune system that allows
life-threatening opportunistic infections and cancers
to thrive.
 The pathogen was identified 2 years after the first
report of the disease in 1981.
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HIV Infection
It infects vital cells in the human immune
system such as
 CD4+ T lymphocytes,
 Peripheral blood mononuclear cells (PBMC),
 Monocytes,
 Dendritic cells,
 Macrophages
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HIV infection leads to low levels of CD4+ T
cells through a number of mechanisms,
 Apoptosis of uninfected bystander cells,
 direct viral killing of infected cells,
 killing of infected CD4+ T cells by CD8 cytotoxic
lymphocytes
When CD4+ T cell numbers decline below a
critical level, cell-mediated immunity is lost,
and the body becomes progressively more
susceptible to opportunistic infections.
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Types of HIV
Two types of HIV have been characterized:
 HIV-1 and HIV-2.
HIV-1 is the virus that was initially discovered is
more virulent, more infective, and is the cause of
the majority of HIV infections globally.
Species
Virulence
Infectivity
Prevalence
Inferred
origin
HIV-1
High
High
Global
Common
Chimpanzee
HIV-2
Lower
Low
West Africa
Sooty
Mangabey
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HIV Genome
 The HIV-1 genome is composed of two identical
copies of single-stranded RNA, consists of nine
genes,
 The high mutation rate during the replication
cycle results in a great likelihood for the
development of drug resistance of the virus.
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HIV Genome
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Clinical Treatment of AIDS
 More than 30 drugs have been developed for
the clinical treatment of AIDS.
 No effective treatment to eliminate the virus
from patients
 Preventive vaccine for HIV would be ideal,
developing a successful preventive vaccine for
HIV is a long and complicated process.
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Antiretroviral medicines
 Nucleoside/nucleotide
inhibitors
reverse
 Non-nucleoside
reverse
inhibitors (NNRTIs),
transcriptase
transcriptase
 Protease inhibitors (PIs),
 Entry inhibitors,
 Integrase inhibitors
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Targets of HIV Therapies
 Multiple proteins involved in the HIV life cycle
have
been
validated
for
antiviral
drug
development.
 Theoretically, each step of HIV replication
could be targeted for anti-HIV therapy .
 The HIV-1 genome consists of nine genes,
which are also important targets for anti-HIV
research.
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HAART
 Highly-Active Antiretroviral Therapy
 The only effective therapy used in clinical
treatment for HIV infection
 A combination of antiviral drugs are used
targeting different steps in the virus life cycle.
 It is used to slow the rate at which HIV makes
copies of itself (multiplies) in the body
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 Why GE-based Therapies
 An increasing number of patients have to face
the problem that, due to the serious drug
resistance, there are no suitable drugs to
choose from, for use in HAART for HIV
infection.
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GE-based Therapies
Genome editing (GE)-based HIV therapy is
 Modification of infection-related genes to produce
HIV resistant cells followed by reinfusion of the
modified cells into patients.
 The ultimate goal is to achieve a functional or
actual cure for HIV infection.
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 In 2009, a patient was functionally cured of HIV
infection by transplantation of allogeneic stem cells
from a donor homozygous for the CCR5 δ32 allele.
 There is a small, but reasonable chance of finding
an human leukocyte antigen HLA-matched donor
homozygous for the CCR5 δ32 allele.
 Mimicking the natural homozygous CCR5 δ32 by
using GE technologies to induce the mutation is a
viable choice.
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The success of the stem cell transplant has
encouraged the continued study of GE-based
HIV therapies.
The general concerns of GE-based HIV therapies
include
 target selection,
 the choice of GE technology,
 and evaluation of safety and efficacy.
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Advances in GE-based Therapies
 Transcription activator-like effector nucleases
(TALENs)
 Zinc-finger nucleases (ZFNs)
 Clustered regularly interspaced palindromic
repeats (CRISPR)/Cas9 systems
The ZFN system--most widely used, there are only
a few reports of GE-based HIV therapies using
the TALEN and CRISPR/Cas 9 systems, these
two GE tools may have great potential.
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ZFNs: Zinc-finger nuclease
 Origin: Eukaryotes
 Compatible with various viral vectors;
 each ZF module (30 amino acids) recognizes
three sequence-specific nucleotides;
 widely used in various cell types and multiple
organisms including humans
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TALENs: Transcription activator-like
effector nuclease
 Origin: Xanthomonas
 TALEs consist of multiple 33–35 amino acid repeats
in which each
nucleotide.
repeat
recognizes
only
one
 TALENs result in targeted double-stranded breaks
which activate DNA damage response pathways.
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CRISPR: Clustered regularly interspaced
palindromic repeats
 Origin: Bacteria
 DNA loci contained in bacterial genomes.
 consists of an array composed of 21–47 nucleotide
(32 on average) short direct repeats with various
intervening spacers.
 is one component of the pathway used by bacteria
to destroy foreign invaders.
 RNA-guided DNA endonuclease; targets multiple
sites simultaneously to produce large gene fragment
deletions.
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The major strategy for GE-based HIV therapies is
 to produce engineered immune cells that are
resistant to HIV infection or replication.
The possible cell types for modification are
 hematopoietic stem cells
 dendritic cells and
 CD4+ T cells
CD4+ T cells and CD34+ stem cells are the two
groups of cells, most frequently used for GEbased HIV therapies.
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Method of GE-based Therapy
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Approaches of GE-based Therapies
The current approaches are mainly classified into
two categories:
 Therapies targeting host genes involved in virus
infection and replication, such as CCR5
 Therapies introducing genes that interfere with
HIV replication, such as host restriction factors
and fusion inhibitors
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Targets of GE-based Therapies
 CD4,
the major receptor for HIV, is very
important for the human immune system,
 Deletion of the CD4 gene is lethal. Several clinical
trials involving CD4 modification for HIV
therapies have been conducted, but these studies
are no longer being investigated for clinical
treatment.
 The CD4 receptor may not be an appropriate
target, the co-receptors for viral entry are ideal
targets for disruption .
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Targets
 CCR5
 CXCR4
 Proviral DNA
 Other targets
 Lens
epithelium-derived growth factor
(LEDGF)
 The mitochondrial translocator protein
(TSPO)
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CCR5
C-C chemokine receptor type 5
 A naturally existing 32 bp deletion in the CCR5
gene leads to a non-functional receptor, which
confers resistance to HIV strains that use CCR5.
 Cells that are naturally resistant to HIV are usually
from donors with the CCR5 d32 genotype
 Multiple methods have been developed to construct
engineered cells with non-functional CCR5
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ZFN-mediated modification of
CCR5
 ZFN-mediated
modification of CCR5 induced
pluripotent stem cells (hiPSCs) and human
embryonic stem cells (hESCs) demonstrated the
potential for treatment of HIV infection
 Mice treated with CCR5-disrupted hematopoietic
stem or progenitor cells (HSPCs) achieved resistance
to HIV infection
 Permanent dysfunction of CCR5 by modification
with ZFN
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Continued…
 In a study, 12 patients with chronic aviremic HIV
infection received a single dose of autologous CD4
T cells modified by ZFN.
 Immune reconstitution and HIV resistance were
evaluated by CD4 T cell count and viral RNA or
DNA detection.
 A significant increase in the CD4 T cell count was
observed and blood HIV DNA levels decreased in
most patients.
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CRISPR/Cas9:
mediated modification of CCR5
 Recently, hiPSCs homozygous for the CCR5 d32
mutation were produced
CRISPR/Cas9 system in
piggyBac technology
using TALEN or
combination with
 These engineered cells showed resistance to
HIV-1 challenge when
monocytes/macrophages.
differentiated
into
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CXCR4:
C-X-C chemokine receptor type 4
Disruption of the C-X-C chemokine receptor
type 4 gene (CXCR4) with ZFN cause
 resistance to T-tropic HIV in several studies
 Human T cells resistance to X4-tropic HIV-1
strains.
 also conferred resistance to X4 HIV-1 in
humanized mouse.
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Continued…
 In another study, disruption of CXCR4 with
ZFNs in CD4+ T cells provided protection
from HIV-1 infection in tissue culture and in
mice.
 After engraftment of engineered T cells, HIV1-infected mice had lower viral load.
 A safe mode of disruption of CXCR4 is still
needed to be discovered.
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Proviral DNA
 Eliminating the HIV proviral DNA
infected cells with GE technologies
from
 The incurability of HIV infection can be
attributed to


the integration of viral DNA into the
human genome and
the persistence of proviral DNA.
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Cleavage by ZFNs
 Up to 80% of integrated viral DNA can be removed
from the human genome with ZFNs targeting the
proviral HIV Pol gene or the whole proviral
genome
 Three ZFNs were designed to cleave the conserved
regions of Gag, Pol, and Rev
 The full-length proviral DNA can also be excised
from human genome with ZFNs targeting
conserved regions within HIV-1 50 and 30 long
terminal repeats (LTRs).
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Cleavage by CRISPR/Cas9
 The LTRs target sites were efficiently cleaved and
mutated by the system.
 The CRISPR/Cas9 system was also able to delete
internal HIV genes from a human chromosome
 Highly
specific targets were identified and
efficiently edited by Cas9 system with guide RNAs
(gRNA) homologous to sequences within the LTR
U3 region of HIV-1
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 HIV-1 infection was prevented in the presence of
multiplex gRNAs in cells expressing Cas9.
 ZFNs have also been designed to target proviral
DNA of other viruses including HTLV-1, HSV-2
and HBV
 Major challenge in clinical treatment is how to
deliver the ZFNs or CRISPR/Cas9 system to the
latently infected cells accurately because these
cells are extremely rare.
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LEDGF
Lens Epithelium-derived Growth Factor
 The first well-characterized cellular cofactor for
HIV-1 integrase.
 It plays a crucial role in viral integration, tethering
the pre-integration complex (PIC) to the nucleus
through interaction with integrase.
 Similarly to the case of CCR5, in CD4+ T cells,
LEDGF depletion is well tolerated.
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Continued…
 In recent studies, all LEDGF expression was
completely eradicated with TALENs.
 The deletion of whole LEDGF gene or the
exons encoding the integrase binding domain
(IBD) resulted in inhibition of viral integration
and impaired virus replication in human cell
lines.
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TSPO:
Mitochondrial Translocator Protein
 was found to inhibit HIV-1 Env glycoprotein
expression
 Although the CRISPR/Cas9 system was only
used to generate a TSPO knockout in 293T cells
for this study, the locus could be an attractive
target for other GE-based strategies
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Co-receptor Usage Shift
Co-receptor usage shift is an important problem in
anti-HIV research.
 CCR5 serves as the predominant co-receptor in
the early stage of HIV infection, and
 CXCR4 plays a key role as co-receptor when
stable infection is established.
HIV co-receptor usage can switch between CCR5
and CXCR4, resulting in failure of protection if
therapies target only one of these co-receptors.
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Concerns in GE-based Therapies
Several issues give cause for concern.
 Safety: is a fundamental concern in GE-based
therapies. A long-term safety evaluation is
needed.
 Efficacy:
 It has yet to be determined if the goal of the
therapy will be to cure the HIV infection or
control the progression of AIDS
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 A technical concern for GE tools is off-target
cleavage events, which can lead to unintended
disruption of physiologically important genes,
and can have unpredictable consequences.
 Predicting possible off-target cleavage sites
and understanding their effects will greatly
improve the efficiency and the safety of GEbased therapies.
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Conclusion
 The current GE-based HIV therapies are mainly
based on reinfusion of modified
lymphocytes or CD34+ stem cells.
CD4+
T
CCR5 is the major current focus because naturally
existing CCR5 d32 confers resistance to HIV
infection,
Production of HIV-resistant cells with an artificial
CCR5 mutation and reinfusing the cells into
patients to confer HIV resistance is the most
common strategy.
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Future Perspective
 Several important scientific problems remain
to be addressed
Co-receptor Usage Shift
 Off-target events
 The
molecular
mechanism
of
the
functional cure is not yet clear.
 With the development of GE technologies and
better understanding of the HIV infection
process, more targets may be approached.

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Reference:
 Gang W.G., 2015. Genome editing-based
HIV therapies. TIBTEC-1229: 1-8
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