News

Can gene editing drive out HIV and hepatitis

viruses from inside cells?
Armed with gene technologies and
CAR-Ts, scientists are attempting
to eliminate viruses that escape
immune detection and lurk in
tissues for years.

By Cormac Sheridan

A
gene-editing therapy based on
CRISPR–Cas9 designed to elimi-
nate HIV-1 infection has rekin-
dled hopes among scientists that
it may be possible to eradicate
the virus and cure the infection. In an in vivo
editing study in non-human primates con-
ducted by scientists from Temple University
and Excision BioTherapeutics, a single dose
of a CRIPSR therapy excised the simian immu-
nodeficiency virus (closely related to HIV-1)
from the DNA of infected animals with no
observable off-target effects or other safety
problems. The equivalent human therapy,
EBT-101, is undergoing a phase 1/2 clinical trial
in people infected with HIV-1.
This CRISPR therapy is part of a wave of An infected lymphocyte with an HIV cluster.
sophisticated genetic approaches targeting
difficult-to-treat chronic infections (Table 1).
In addition to gene editing, therapies, (HBsAg). “The virus can hide in this blizzard California, San Francisco. They tested T cells
including antisense oligonucleotides, short of protein and the immune system has a very modified ex vivo to attack HIV-1-infected CD4
interfering RNA (siRNA), gene therapy, thera- hard time finding it,” says Melanie Paff, vice T cells, employing a retroviral vector encod-
peutic vaccines and chimeric antigen receptor president and medicine development leader ing a CAR that consisted of the extracellular
(CAR)-T cell therapies, are being brought to on GSK’s HBV program. This excessive immune and transmembrane domains of the CD4 T cell
bear on a range of pathogens, but most promi- stimulation results in T cell and B cell exhaus- receptor linked to the cytoplasmic domain of
nently on HIV-1 and chronic hepatitis B virus tion, leading to liver fibrosis, cirrhosis and the ζ chain of the CD3 T cell receptor. “Those
(HBV) infection. cancer. Nucleoside or nucleotide analogue cells could persist over long periods of time,
Once a person is infected and after the therapy can lower but not eliminate the risk but they did not actually impact the viral load,”
acute stage resolves, these viruses can persist of liver fibrosis and cirrhosis. says Boro Dropulić, a scientist-entrepreneur
indefinitely — in CD4 T cells, macrophages and Although most people link the use of modern who is executive director of Caring Cross. They
microglia, in the case of HIV-1, and in hepato- genetic technologies with cancer, auto­ lacked co-stimulatory domains to boost their
cytes, in the case of HBV. Although antiretro- immune disease and genetic disease, some expansion and activity and were themselves
viral drugs keep HIV-1 replication in check, as of these tools have long histories in infectious susceptible to HIV-1 infection.
soon as treatment is interrupted, the infection disease. In fact, CAR-T cell therapies were ini- Dropulić had already attempted to develop
quickly rebounds from these viral reservoirs. tially developed for tackling HIV-1 infection a curative HIV-1 therapy with VIRxSYS, which
In chronic HBV infection, covalently closed rather than cancer. The first clinical trial began he founded. He collaborated with June and
Credit: Phanie / Alamy Stock Photo

circular DNA molecules persist in the nuclei
of liver cells and drive viral replication. In
addition, some viral DNA sequences do not
circularize or replicate but instead integrate
into the host genome, producing massive
quantities of non-infectious viral protein par-
ticles that contain hepatitis B surface antigen
more than two decades ago to tackle HIV-1
infection with CAR-T cells. It was conceived
by a team including Kristen Hege at the bio-
tech firm Cell Genesys and academic scientists
and clinicians, including Carl June, then at the
National Institute of Allergy and Infectious
Disease, and Steven Deeks of the University of
Deeks in testing an ex vivo gene therapy com-
prising CD4 T cells transformed with a lenti-
viral vector — the first to be tested in humans
— encoding an antisense molecule that blocks
expression of the HIV envelope gene. This,
too, proved to be ineffective, but Caring Cross,
which he co-founded with scientific director

nature biotechnology
News

Table 1 | Selective therapies in development for HIV-1 and chronic HBV infection

Developer Therapy Mechanism Indication Clinical stage

GSK, Ionis Pharmaceuticals Bepirovirsen Antisense oligonucleotide with a 2′-O-methoxyethyl gapmer Chronic HBV Phase 3
design, which suppresses production of all HBV proteins by infection
binding a sequence present in all HBV mRNA molecules
Vaccitech (Oxford, UK) VTP-300 Prime–boost therapeutic vaccine comprising Chronic HBV Phase 2
replication-defective chimpanzee adenovirus infection
(ChAdOx1-HBV) and modified vaccinia Ankara (MVA-HBV),
both encoding the inactivated polymerase, core and
complete S region from an HBV genotype C consensus
sequence
Replicor (Montreal) REP 2139-Mg Nucleic acid polymer that disrupts the formation of Chronic HBV Phase 2 and
HBsAg-containing subviral particles infection compassionate
access program
Arbutus Biopharma Imdusiran (AB-729) N-Acetylglucosamine-conjugated siRNA designed to block Chronic HBV Phase 2
all HBV transcripts infection
Roche, Dicerna RG6346 (RO7445482) GalXC siRNA molecule that targets HBsAg mRNA Chronic HBV Phase 2
infection
Vir Biotechnology, Alnylam VIR-2218 (ALN-HBV02) N-Acetylgalactosamine-conjugated siRNA directed at a Chronic HBV Phase 2
Pharmaceuticals sequence in the HBV genome that is present in all transcripts infection
Caring Cross DuoCAR T cell therapy Autologous T cells transduced ex vivo with a lentiviral vector HIV Phase 1/2a
encoding a CAR directed against two HIV gp120 epitopes
Excision BioTherapeutics EBT-101 Intravenously delivered AAV9 encoding Staphylococcus HIV Phase 1/2a
aureus (sa)Cas9 endonuclease and two guide RNAs that
lead to removal of three large segments of chromosomally
integrated proviral HIV DNA
Shanghai BDgene BD-111 mRNA-based CRISPR–Cas9 editing components, delivered Refractory herpetic Phase 1/2
Therapeutics (Shanghai) by corneal injection, designed to clear herpes simplex virus 1 viral keratitis
(HSV-1) infection
SCG Cell Therapy (Singapore) SCG101 Autologous cytotoxic T cell receptor T cell therapy directed HBV-related Phase 1/2
against HBsAg hepatocellular
carcinoma
Sources: ClinicalTrials.gov, PubMed, company websites

Rimas Orentas as a not-for-profit to develop
sustainable, curative immunotherapies, is
building on that experience with lentiviruses
in developing a modern CAR-T therapy. Deeks
and Mehrdad Abedi, of the University of Cali-
fornia, Davis, are clinical collaborators.
Their anti-HIV duoCAR-T cells are designed
to rapidly sense and kill infected cells. The
duoCAR-Ts are genetically engineered with
two CARs in a single cell, each recognizing
a different conserved epitope on the HIV-1
glycoprotein envelope (gp120). The mode of
action is two-pronged: the cytotoxic CAR-T cell
receptor targets and kills HIV-1-infected cells
while simultaneously protecting the CAR-T
from infection, and the second receptor acts
as a viral fusion inhibitor.
The duoCAR-T cells will not directly attack
latently infected cells, however, as these do
not express gp120 protein on their cell sur-
face. “We may need other mechanisms in
order to activate that truly latent population
that does not express gp120,” says Dropulić.
Data from the study will shed valuable light
on this question, as the trial participants will
stop taking their HIV medications until viral
rebound occurs.
Approaches that target latently infected
cells directly are also in development. For
example, a group led by Karin Metzner of the
University of Zurich and Zurich University Hos-
pital, Switzerland, recently described a highly
specific ‘shock-and-kill’ strategy designed to
kill latently infected cells. The therapy com-
bines a CRISPR-guided transcription activa-
tion domain — to initiate transcription of HIV-1
genes — with the truncated Bid (tBid) suicide
gene system, which is conditionally activated
by HIV-1 accessory proteins. In initial cell cul-
ture studies, the system was able to kill about
50% of latently infected cells. “At this stage, it
would not be a cure,” says Metzner.
Attaining a 100% kill rate in a clinical set-
ting is unlikely, but the aim is to improve this
parameter. “No one really knows to what
extent you have to reduce this reservoir,”
says Metzner. “I’m convinced we don’t need
to eliminate the reservoir entirely.” A clini-
cally meaningful outcome would be one that
matches what is observed in people known as
elite controllers, who manage to suppress the
virus in the absence of antiretroviral drugs.
The shock-and-kill approach is ripe for
further optimization. Its cell-killing compo-
nents are currently delivered in two adenovi-
rus vectors, which are modified to transduce
CD3-expressing T cells, but Metzner’s team
aims to replicate the system in a single
‘gutless’ adenovirus vector with a large
payload capacity before embarking on animal
studies. Her co-author and collaborator
Andreas Plückthun, also of the University of
Zurich, has pioneered its development.
In the meantime, data from Excision’s trial
of EBT-101 will provide important insights
into feasibility of using a simpler, more direct
approach. The therapy, which is delivered in an
adeno-associated virus 9 (AAV9) vector, is not
designed to reactivate latent virus but to elimi-
nate the possibility of reactivation occurring.
It is not the first attempt to use gene editing
against HIV-1. Almost a decade ago, Carl June’s
group used zinc finger nucleases to disrupt
the C-C chemokine receptor type 5 (CCR5)
locus in the T cells of people with HIV-1. (CCR5

nature biotechnology
News
encodes a co-receptor required for HIV-1
entry.) Viral rebound occurred after antiviral
drug interruption, however. Excision’s CSO, TJ
Cradick, says the multiplex editing capability
of EBT-101 — its two guide RNAs target three
locations on the HIV-1 genome — adds another
layer of protection by minimizing the risk of
escape variants emerging.
How potent this CRISPR-based therapy
will turn out to be will depend on its ability
to reach the anatomical sites where the latent
cell reservoirs reside, including the lymphoid
tissues, the bone marrow and the brain,
and whether it can transduce the latently
infected cells with high efficiency. Even so, it
may not be necessary to target and eliminate
the virus from every one of the infected cells
for the approach to be effective. The situation
is substantially different from that in genetic
disease, says Cradick; in that case, every cell
carries the target sequence, which increases
the risk of off-target edits.
For chronic HBV infection, a cure may
be much closer than for HIV-1 infection.
London-based GSK expects to report data
from two phase 3 trials of its antisense mole­
cule bepirovirsen in 2025. The goal of therapy
is threefold, says Paff: to block viral HBV
repli­cation, eliminate HBsAg production and
restore immune system function. Bepiro-
virsen, GSK claims, does all three, by target-
ing pre-genomic RNA to stop viral replication,
by blocking viral mRNA transcription to pre-
vent HBsAg production, and by activating
the innate immune system through a Toll-like
receptor 8 agonist activity.
In an earlier phase 2b trial, bepirovirsen
led to clearance of both HBsAg and HBV in
9% of patients who were on nucleoside or
nature biotechnology
nucleotide analogue therapy and 10% of
patients who were not. This effect was main-
tained 24 weeks after they stopped taking the
antisense molecule. Some patients fared bet-
ter than others, however. “We found this very
interesting predictor of response,” Paff says.
“It depends on how high your surface anti-
gen is to begin with.” For those with a baseline
plasma level of HBsAg below 3,000 interna-
tional units (IU) per milliliter, the response
rates for patients on or not on nucleoside ana-
logue therapy were 12% and 25%, respectively.
The two phase 3 trials have recruited patients
who match this criterion.
To improve the responses further, GSK
is also investigating combinations of bepiro-
virsen with immune enhancers, such as
peginterferon (data from a phase 2b trial
are imminent) and a therapeutic vaccine it is
developing. Many other firms are developing
siRNA-based therapies, which have substan-
tially lowered levels of HBsAg. “In the natural
history of hepatitis B, a low HBsAg concen-
tration is associated with a better prognosis,
when it’s achieved naturally, by immune con-
trol,” says Geoffrey Dusheiko, emeritus profes-
sor of medicine at University College London.
“We’re not sure if knockdown of HBsAg by
small interfering RNA and antisense oligonu-
cleotides will have the same effect, but I think
that is encouraging, to continue to pursue
functional cures.” However, “a complete steri-
lizing cure does not seem to be obtainable at
the present time,” he says, given the challenge
of eradicating all HBV integrants.
Other approaches face the same challenge.
GigaGen, a subsidiary of Barcelona-based
Grifols, aims to file an investigational new
drug application next year, ahead of an initial
trial of its recombinant polyclonal antibody
therapy for chronic HBV infection. It gener-
ates high antibody diversity by capturing large
numbers of B cell repertoires from immunized
individuals in a microfluidics system. “We’re
not engineering the antibodies, but we can
choose which antibodies go into our mix and
which ones do not,” says CEO Carter Keller.
“It creates an incredibly potent therapy.”
It is about a thousand-fold more active than
conventional plasma-derived hyperimmune
globulin, he says, and the company believes
it can clear the virus rather than just suppress
its replication.
Gene-editing efforts to eliminate HBV are
also underway. Beam Therapeutics recently
reported that its adenine base editor reduced
HBV viral DNA by 98% while achieving sus-
tained 3–4 log reductions in HBsAg levels
after a single injection into a mouse model.
Precision BioSciences has reported dura-
ble HBsAg reductions of 96% in a mouse
model following treatment with its homing
endonuclease therapy, which is based on
an enzyme isolated from the algal species
Chlamydomonas reinhardtii.
Meanwhile, the list of patients cured of
HIV-1 infection following a transplantation of
CCR5-deleted hematopoietic stem cells for
cancer, though small, is slowly growing with
another addition. The procedure is potentially
curative but also risky, costly and difficult to
scale. Developers of these high-tech thera-
pies will also need to face these issues as they
tackle two viruses that continue to have severe
impacts on public health.
Cormac Sheridan
Dublin, Ireland