ISTH 2022 Congress Review

ISTH 2022 Congress Review
Thinking Differently in Haemophilia:
Gene Therapy at ISTH 2022
Welcome to the ISTH 2022 Congress Review.
The ISTH is dedicated to advancing the understanding,
prevention, diagnosis, and treatment of conditions related to
thrombosis and haemostasis – and has over 7,000 clinicians,
researchers, and educators working together to improve the
lives of patients around the world.
For this review, we have selected abstracts and presentations
with a focus on gene therapy for people with haemophilia. This
includes new vector shedding data from GENEr8-1 [page 5],
and a QoL analysis from HOPE-B [page 6]. We also bring you
a wealth of new information and advances in gene therapy
technology and engineering, offering a tantalising glimpse
of the novel therapies that could be next over the horizon.
I hope you enjoy our summary of the key data.
Professor Charles Hay
MORE INSIDE:
Clinical data: Gene therapy for haemophilia A page 4
Clinical data: Gene therapy for haemophilia B page 6
Immunogenicity of gene therapy page 8
Factor replacement and other therapies page 18
The WFH GTR
Long-term follow-up
for gene therapy.
Read more on page 2
Evolving clinical practice
International accreditation
and safe-guarding quality.
Read more on page 2
The future of gene therapy
Advances in engineering
and gene editing.
Read more on page 9
Bleeding tendencies
in haemophilia
Understanding the
bleeding phenotypes.
Read more on page 16
The poster presentations and
abstracts can be found here
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ISTH 2022 Congress Review
Long-term follow-up for gene therapy
Registry collaborations are an effective way to pool enough data to enable
robust evaluation of rare AEs and durability of gene therapy. Linking
registries is an extensive process involving many steps and collaboration
among multiple partners.
The WFH GTR is a prospective, observational,
and longitudinal registry. The core data set
was developed through an iterative review
process by the GTR Steering Committee.
To maximise the utility of data in existing
registries – and avoid the need for duplicate
data entry at individual HTCs – the WFH GTR
includes a data integration programme.
Collaboration has already begun with the
American Thrombosis and Hemostasis
Network, HemoNed, and the Canadian
Bleeding Disorders Registry. Common data
definitions were set, and careful comparison
of data field measurements conducted to
facilitate the technical mapping of data fields
between the GTR and partner registries;
de-identified data will now be transferred
to the GTR on a regular basis. The GTR and
partner registries will help establish longterm
follow-up as the standard of care for
gene therapy, and discussions on potential
integrations with other national registries are
ongoing. The WFH GTR will ensure that rare
AEs will be detected in a small population
over a large geographical area. The GTR will
also help further establish long-term followup
as the standard of care for gene therapy.
[PB0212]
possibilities, including gene therapy. A tight
network of centres structured along a huband-spoke-model
will address all aspects of
gene therapy, from dosing and surveillance in
the immediate post-infusion period, to longterm
follow-up. The content and logistics of
the multidisciplinary auditing and certification
procedures are in development, and it is
expected that the auditing process will begin
in 2023. [PB1127]
In a session on health literacy in the nurses’
forum, there was a presentation outlining the
development of a new educational tool for use
on the ISTH website. The role of the nurse in
haemophilia care is to educate, guide, and
support patients. The advent of gene therapy
is likely to create a new class of patient who
has shifted from a severe to mild phenotype,
and who will therefore need a different
management approach. In the future, the
role of the haemophilia nurse is likely to
require a greater emphasis on psychosocial
support for gene therapy patients. Inherently,
there are challenges for incorporating gene
therapy into clinical practice, with issues of
patient information, selection, education,
and consent. There are also practical
considerations for preparation, storage,
and administration, as well as the need for
monitoring and laboratory investigations.
Communication with patients and caregivers
remains key. The new ISTH educational
tool is designed for nurses and includes an
introduction to gene therapy for haemophilia
– from basic terms and concepts to trial data
and frequently asked patient questions. The
tool uses an adaptive e-learning approach
to help contribute to a holistic approach and
to promote the acquisition of knowledge.
Further e-learning modules available on the
site include emerging nonfactor approaches
for people with haemophilia. [NUR 02]
Haemophilia: Evolving clinical practice
The Changing Role of Nurses
Role of nurses shift
from teacher to coach
Gene therapy represents a fundamental shift in the treatment of haemophilia,
but the approach differs significantly from all other current treatment
protocols and presents a challenge for both medical staff and centres.
New normal:
Patients adjusting
to a mild phenotype
Inform the patient and
explain their treatment
The international accreditation of haemophilia
centres in Europe is run by EAHAD and EHC.
Centres are designated as EHCCC or EHTC
based on specific requirements that evaluate
their ability to provide care for haemophilia
and allied disorders. Boban et al. presented a
poster on the novel accreditation of European
haemophilia treatment and gene therapy
centres. EAHAD, in collaboration with EHC,
have taken steps to define measures to
safeguard and improve the quality of care for
bleeding disorders care throughout Europe,
to build a novel model for implementation of
gene therapy, and to provide a new auditing
and certification protocol. Haemophilia
centres are organised to offer multidisciplinary,
integrated, and comprehensive
care – as well as equal access to all treatment
More visits
with a new treatment
vs standard of care
Requires
confidence and trust
in the body and mind
React and treat
vs wait and see
Be present at all times
and explore how the
patient is feeling
Mulders-van der Meer [NUR02]
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ISTH 2022 Congress Review
Clinical data: Gene therapy for
haemophilia A
Several gene therapies in development continue to collect efficacy, safety,
and QoL data after a single infusion in people with haemophilia A.
Haemostatic results for up to 6 years
following treatment with a single infusion of
ROCTAVIAN ® (valoctocogene roxaparvovec)
were presented by Laffan et al. ROCTAVIAN ®
is indicated for the treatment of severe
haemophilia A in adult patients without a
history of factor VIII inhibitors and without
detectable antibodies to AAV5. Please read
the ROCTAVIAN ® SmPC before prescribing.
In the Phase 1/2 trial, all participants showed
sustained improvements in FVIII activity, ABR,
and use of exogenous FVIII. Updated, detailed
safety and efficacy assessments from 6-year
follow-up data from the 6x10 13 vg/kg cohort
and 5-year follow-up data from the 4x10 13 vg/kg
cohort were shared, showing a sustained
reduction in treated bleeds in both groups.
Haemostatic efficacy was maintained for
12 of 13 participants over the study period,
and the 7 participants dosed with 6x10 13 vg/kg
demonstrate ongoing haemostatic efficacy
without routine prophylaxis 6 years postgene
transfer. In the 4x10 13 vg/kg cohort,
one participant resumed FVIII prophylaxis
for 1 month during Year 5, and is currently
using on-demand treatment, with no need
for FVIII infusions over the past 20 weeks.
Mean annualised FVIII infusion rate over entire
study observation period remained low, with a
96% and 93% reduction from baseline in the
6x10 13 and 4x10 13 vg/kg cohorts, respectively,
and previously observed trends regarding
change in FVIII activity were maintained.
Mean QoL was maintained (4x10 13 vg/kg
cohort) or improved (6x10 13 vg/kg cohort) over
the study period, with individual variation. With
regards safety, there was one event of parotid
acinar cell carcinoma – but genomic analyses
supported initial assessment of this event
as unrelated to valoctocogene roxaparvovec.
Overall, the safety profile remains unchanged
from previous reports. [OC 21.2]
Liu et al. presented comparative effectiveness
of valoctocogene roxaparvovec and FVIII
prophylaxis, estimated through propensity
scoring. The aim of this post-hoc analysis
was to compare bleeding outcomes among
adults with severe haemophilia A treated
with valoctocogene roxaparvovec versus
prophylactic FVIII replacement. Data from
112 participants who rolled over into GENEr8-1
were compared to 73 participants enrolled
in a prospective noninterventional study who
were negative for FVIII inhibitors, anti-AAV5
antibodies, and HIV. Comparable cohorts
were generated based on propensity scores
using standardised mortality ratio weighting
to weight the control cohort to match baseline
characteristics of the intervention cohort.
The analysis found that mean treated and
all-bleeds ABR were significantly lower in
the intervention versus control cohort, with
an absolute difference of –3.6 (P

ISTH 2022 Congress Review
Gonen-Yaacovi et al. showed the design for a
clinical study of ASC618 – a highly potent and
minimally sized vector containing a hepatic
combinatorial bundle promoter, liver-specific
codon optimisation, and a highly expressing
bioengineered human FVIII (ET3) transgene,
designed to express FVIII protein for the
treatment of severe and moderately severe
haemophilia A. In preclinical studies, ASC618
was well tolerated at all doses evaluated with
Clinical data: Gene therapy for
haemophilia B
no toxicologically significant microscopic
findings. Now, the safety, tolerability and
preliminary efficacy will be assessed in a
prospective clinical study. The decision of
dose escalation versus dose expansion in
each cohort will be based on FVIII levels
and safety evaluations. Following infusion,
patients will be closely monitored for FVIII,
ALT, and AST for up to 5 years post-infusion.
[PB0662]
Breakthrough bleeding still occurs with many replacement therapies.
Achieving FIX levels after gene therapy is expected to normalise haemostasis
in people with haemophilia B.
Steve Pipe presented the abstract from
Itzler et al. on HRQoL data from the Phase
3 HOPE-B trial. Fifty-four adults with
haemophilia B (FIX ≤2%) received standard
of care FIX concentrate prophylaxis in the
6-month lead-in period followed by a single
infusion of etranacogene dezaparvovec.
Quality of life was a prespecified secondary
endpoint, and HRQoL was assessed with the
haemophilia-specific Hem-A-QoL during
lead-in, and again at 6 and 12 months –
alongside other patient-reported outcomes.
The results showed significant modelbased
mean differences in both scores
and percentage improvement compared
with the lead-in period, including a
total score change of –5.50 (P

ISTH 2022 Congress Review
Immunogenicity of gene therapy
Immune responses to gene therapies have been associated with infusion
reactions, liver enzyme elevations, and loss of transgene expression.
The predominant AE in clinical trials of
AAV-based gene therapy for haemophilia
has been ALT elevation. Although capsiddirected
cytotoxic T-cell responses have
been reported, the underlying mechanisms
remain unclear. Batty et al. shared data on
innate and adaptive immune responses to
AAV gene therapy in a canine model. Severe
haemophilia A dogs (N=10) were treated
using one of three AAV5-BDD FVIII constructs
with a hybrid liver promoter (AAV5-HLPcFVIII).
Capsid immune responses were
evaluated using total antibody and ELISPOT
assays, and serum innate immune responses
were evaluated using a cytokine array and
complement function. Liver biopsies were
used to quantify vg copies and cFVIII mRNA
at baseline and 3 months. The results showed
FVIII expression for the 7 dogs treated with
codon-optimised constructs. Dose response
was seen at 3 months, with higher one-stage
FVIII:C in the high-dose compared to lowdose
cohort. Liver biopsies demonstrated
AAV5-HLP-cFVIII in all animals, with
correlation between vg and cFVIII mRNA
copies. Anti-AAV5 capsid antibodies were
detected starting Day 7 post-treatment,
and no new FVIII inhibitors were detected.
The dogs displayed variable serum cytokine
profiles, with no change in mean levels
24 hours post-dosing, and no blood
biomarkers of innate immune activation
detected. Higher baseline and post-dose
levels of TNF-alpha, IL-2, IL-6, IL-7, IL-15,
and IL-18 were observed in the dog with a
FVIII inhibitor history. No capsid-specific
T-cell responses or complement activation
was detected. Further liver studies
are ongoing. [OC 01.5]
Chapin et al. gave the results of a
translational analysis of immune components
in peripheral blood from severe haemophilia
A patients treated with TAK-754 (formerly
BAX 888) – an AAV8 vector with a
codon-optimised BDD FVIII transgene.
Cell-mediated cytotoxic T cell responses
in peripheral circulation have been reported
in clinical trials but have not consistently
correlated with transaminase elevation
and loss of transgene expression. The
aim of this analysis was to report clinical
biomarkers of immunogenicity and present
an immunogenicity transcriptomics analysis
from peripheral blood of treated patients.
Results showed no changes in cytokines
within 24 hours of infusion. Transaminase
elevation and decline of FVIII expression
occurred 4–9 weeks post-infusion despite
use of glucocorticoids. No significant changes
in complete blood count populations were
observed. ELISpot assays did not correspond
with loss of FVIII transgene expression or
transaminase elevation, and bulk mRNA
transcriptomic analysis did not demonstrate
significant changes in dendritic or natural
killer cells, NF-κB, IL-6, TLRs 1–8, or T cell
pathway signals. Furthermore, a small
transient increase in TLR9, TNF-alpha, CCL5,
and IRF7 signals occurred 8 hours after
infusion without activating a Type 1 interferon
response. The authors concluded that
TAK-754 evokes a limited immune response
as measured in blood – demonstrating that
such methods have limited value for clinical
decision-making or post-hoc analysis.
Efforts to optimise AAV gene therapy should
focus on targeting tissue-specific analysis
and improving preclinical immunogenicity
models. [PB0211]
It has previously been suggested that
elimination of N-glycosylation in C1 domain
reduces the immunogenicity of gene therapy,
and that a glycoepitope may exist in the
C1 domain. Fan et al. examined the impact
of FVIII glycosylation following AAV-mediated
gene therapy, with the aim of further
characterising the immunogenic glycoepitope
The future of gene therapy
region. Haemophilia A mice were injected
with AAV-FVIII and AAV-N2118Q, followed by
challenge with repeated FVIII injections. FVIII
expression was initially detected in all treated
mice for 8 weeks; however, this subsequently
dropped to very low or undetectable at Week
12 in the BDD group but was only slightly
decreased in the N2118Q group. Following
FVIII challenge, the N2118Q-treated mice
showed higher resistance to inhibitor
formation than those in the BDD group.
Higher proliferation rates or cytokine levels
were detected in mouse cells and human
PBMCs isolated from inhibitor subjects
cultured with MP1 and MP2, but not with MP3
or non-mannosylated peptides. Cells from
non-inhibitor control subjects did not exhibit
increased proliferation when stimulated with
any peptides. [OC 47.5]
Gene therapy is in its infancy, and many new approaches are in development
to enhance and improve the delivery and specificity of transgenes.
In a state-of-the-art session, Elena
Barbon presented on engineering of AAV
to enhance cell-specific transduction.
AAV are commonly used viral vectors for
gene therapy since they are replication
defective, but despite promising clinical
results, challenges remain for AAV-directed
gene therapy, including immunity to the
virus in the general population, which may
affect the widespread applicability of AAVbased
therapeutics. There are also questions
around re-injection of the vector, and dilution
in paediatric patients. Additionally, the
platform cannot accommodate very large
transgenes. There is a need to improve the
AAV vector to give improved transduction
efficiencies, which may ultimately allow
a lower dose, and consequently result
in lower immunogenicity, and reduced
neutralisation by pre-existing antibodies. Key
engineering strategies include site-directed
mutagenesis, capsid shuffling, and peptide
insertion. Dr Barbon showed data from
AAV2 vectors devoid of multiple surfaceexposed
tyrosine, suggesting efficient
transduction and correction of phenotype in
haemophilia B mice, as well as results from
experiments with capsid shuffling technology.
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ISTH 2022 Congress Review
The conclusions noted that AAV capsid
engineering – together with rational design
of vector genomes – are valuable strategies
that may be able to address several issues
with gene therapy. A challenge remains in
translating the results, due to differences in
serotype, cell types, and the in vivo models
used for validation. [SOA 15.1]
Peter Lenting gave a talk on the use of
nanobodies to enhance AAV targeting. One
possible use may be in the purification of
AAV particles. The advantage is that this is
a one-step purification, with yield greater
than 70%. However, the nanobodies cannot
distinguish between empty and loaded
virions, and are more expensive than other
purification techniques. Also, it is unknown
how the AAV affinity for nanobodies might
affect the transgene. Since not all cell types
can be infected with AAV, and off-target
transduction may have unexpected results,
nanobodies may improve cell-specific
targeting. There are two proteins on the
AAV capsid in a ratio of 1:10, and which are
encoded by a single gene. To improve cellspecificity,
it is possible to knock out one of
the AAV binding sites and introduce a new
one by leveraging a non-functional part of
the protein loop. A further advantage is that
nanobodies are very small, so they do not
increase the size of the transgene beyond
the vector capacity. Nanobodies are also
characterised by low immunogenicity and
can be expressed as intracellular proteins
(intrabodies) – which opens up the possibility
of intracellular targets. [SOA 15.2]
Tonetto et al. presented on base- and
prime editing of DNA as a new therapeutic
option for haemophilia A. Recently, DNA
base- and prime editing approaches that
do not rely on HSR have been developed
to cleanly install or revert point mutations.
The group used cellular models created by
expression of recombinant FVIII variants.
The aim was to correct representative F*
missense and nonsense variants at the DNA
level, thus rescuing FVIII expression. The
results showed that in transient and stable
expression systems, c.6046C>T(p.R2016W),
c.6496C>T(p.R2166*), c.6545G>A(p.R2182H),
c.6682C>T(p.R2228*), and c.6683G>A(p.
R2228Q) led to reduced secreted FVIII
levels, while the p.R2228* change produced
appreciable FVIII protein levels attributable to
a truncated isoform. Screening of large sets
of base- and prime edited variants and guide
RNAs identified combinations that rescued
FVIII secretion for the p.R2166, p.R2182H,
and p.R2228Q mutations. Extensive studies
on the p.R2166* and p.R2228Q changes
demonstrated that the reversion of mutations
– detectable at DNA levels – resulted in
appreciable rescue of secreted FVIII protein
and activity levels (up to 20% of FVIII-WT).
This is the first time that a base- and
prime editing approach has been applied to
haemophilia A and provides the proof-ofprinciple
for the efficacy of base editing in
cellular models. Further studies in BOECs
are currently in progress, and will lay the
foundation for studies in animal models
– which might open a new avenue toward
an innovative and personalised cure for
haemophilia A. [OC 01.1]
Ultrasound-mediated gene delivery with
microbubbles is a potentially effective
method of non-viral gene delivery to treat
haemophilia A. Previous studies have found
LSECs could be targeted at a lower power
than hepatocytes. Lawton et al. hypothesised
that gene expression with lower energy
conditions would be comparable to higher
energy conditions, but with less damage to
the liver or induction of anti-FVIII inhibitors.
Mouse liver was injected via the portal vein
with a combination of plasmid DNA and
microbubbles. Simultaneously, a pulsed
therapeutic ultrasound transducer was
applied to the liver surface for 1 minute
at 1.1 MHz frequency and 14 Hz PRF.
Haemophilia A mice were separated into
two different ultrasound condition groups:
low energy targeting predominantly
endothelial cells, or high energy targeting
predominantly hepatocytes. A highexpressing,
endothelial-specific hFVIII
plasmid (pUCOE-ICAM2-hF8/N6-X10) was
used. FVIII activity levels for both energy
groups stabilised around 10% at 84 days
post-treatment. Fluorescent staining showed
co-localisation of hFVIII and Lyve-1 at Days 7
and 120. Transaminase levels indicated the
low energy group had lower transient liver
damage than the high energy group in the
first week, with both groups returning to
baseline by Week 2. These findings show that
LSECs can be targeted for transfection using
lower energy than previously established, and
that this approach can produce FVIII activity
over 84 days. The ability to target different
cell types with ultrasound-mediated gene
delivery may be useful in a multitude of
genetic conditions. [OC 12.5]
Among all mutations causing haemophilia
A, those affecting splicing are relatively
frequent – particularly in severe forms.
These mutation types, often leading to
exon skipping, can be rescued by RNA
therapeutics based on variants of the key
U1snRNA spliceosomal component, as
shown in several human disease models.
Peretto et al. discussed the rescue of an FVIII
splicing variant with engineered U1snRNAs
in two brothers with moderate haemophilia
A. Bioinformatic analysis did not predict an
impact on splicing for the F8 c.1752+5g>c
mutation; however, splicing pattern analysis
of the hepatoma cells expressing the
pIVS11+5c variant revealed that the change
leads to exon 11 skipping, with low levels
of correctly spliced transcripts (~10 %).
Co-transfection of U1snRNA variants
improved FVIII exon 11 definition and thus
inclusion, with the compensatory U1snRNA
associated with exon 11 inclusion up to 92%.
This study provides experimental evidence
that the c.1752+5g>c mutation impairing
F8 exon 11 leads to exon skipping with trace
levels of correct transcripts. Importantly,
the defective exon can be restored with
appropriately designed U1snRNA variants,
which are currently under investigation
through lentiviral-mediated delivery in BOECs
isolated from patients with haemophilia A.
[OC 21.1]
Treatment of canine haemophilia A via
intraosseous delivery of an FVIII lentiviral
vector was showcased by Rementer et al.
The vector – incorporating a platelet-specific
promoter Gp1bα and canine FVIII gene – was
injected into the tibia or iliac bones of four
dogs with haemophilia A. Prior to injection,
the dogs were treated with an immune
modulation regimen to minimise immune
response. Following the procedure, blood
samples were taken at various timepoints.
All dogs recovered well from the procedure
and had blood chemistry values within
normal ranges. Expression of cFVIII was
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ISTH 2022 Congress Review
examined in platelets and plasma by ELISA
and aPTT assays. Canine FVIII was detected
in platelets, with the highest expression at
5–10 mU/10 8 platelets around 1–2 months
post-procedure. Expression persisted for
the duration in all treated dogs. FVIII was
not detected in the plasma following perioperative
delivery, indicating that it is not
released into the plasma. The correction of
the haemophilia phenotype was evaluated
by WBCT, which was shortened in multiple
time points soon after intraosseous gene
therapy, indicating improved haemostasis.
Furthermore, the therapy was well tolerated
and did not produce any toxicity, as evaluated
by CBC and blood chemistry analysis,
and only one animal developed anti-FVIII
inhibitors, which decreased over time.
Encouragingly, the dogs experienced fewer
bleeding events per year after gene therapy
treatment compared with the baseline
prior treatment. This study demonstrates
a potential strategy for safe and effective
application of gene therapy in vivo for treating
people with haemophilia A. [OC 21.3]
Lima et al. shared their work on the
development of a viral vector for haemopexin
protein expression for gene therapy in
disorders associated with increased levels
of free extracellular heme. Haemopexin is
responsible for removing free heme from
the circulation and has been shown to be
depleted in haemolytic diseases. An AAV-
8 vector was produced containing the full
cDNA sequence of human haemopexin; this
was injected into mice with escalating doses
of 2×10 12 , 1×10 13 and 2×10 13 vg/kg, while
a control group received an empty vector.
Expression was confirmed in liver samples
obtained after 6 weeks, and functional
activity assessed by heme challenge and
phenylhydrazine-induced haemolysis.
Overall, sustained expression of human
haemopexin was observed up to 58 weeks
in plasma. Expression was dose-dependent
and not associated with clinical signs of
toxicity. Levels of human haemopexin were
significantly reduced by heme infusions and
phenylhydrazine-induced haemolysis. There
was no clinical toxicity or laboratory signs of
liver damage in these preliminary short-term
studies of heme challenge. Additional studies
are required to confirm the biological effect of
the transgenic protein and its effect in animal
models of chronic haemolysis. [PB0729]
Due to the packaging constraints of the
AAV vector, minimised promoter elements,
such as hAAT/ApoE and TTR, have been
developed to direct FVIII expression to the
liver. Lindgren et al. presented their work to
determine the cellular specificity of hAAT/ApoE
and TTR promoters after systemic delivery
of AAV8 vectors. AAV8-TTR-GFP (n=4)
and AAV8-hAAT/ApoE-GFP (n=3) were
administered to HA-CD4KO mice, and
LSECs and hepatocytes analysed for GFP
expression by flow cytometry. The results
showed AAV8-TTR-GFP produce GFP in a
similar proportion of hepatocytes and LSECs.
Delivery of AAV8-hAAT/ApoE-GFP also
resulted in GFP expression in both cell types;
immunofluorescent staining confirmed the
flow cytometry data. The findings suggest
AAV8 constructs under control of minimised
hAAT/ApoE and TTR promoter elements drive
transgene expression in both hepatocytes
and LSECs. Further studies are required to
understand the biological consequences of
FVIII expression in hepatocytes and LSECs
after AAV-mediated gene therapy. [OC 12.1]
New approaches in development:
Gene editing
Despite phenotypic improvement in clinical trials, universally sustained
AAV-mediated Factor expression at levels necessary to eliminate bleeding
has not been achieved, and many new approaches are in development –
including gene editing.
Sarangi et al. reasoned that a gene-editing
strategy to introduce coagulation FVIIa into
the host genome may augment haemostasis
and alleviate the phenotype in haemophilia.
A novel AAV vector was designed, containing
mFVIIa to target the murine Rosa26 locus
using CRISPR/Cas9-mediated gene editing.
The novel triple vectors designed for
expression of guide RNA, Cas9, and mFVIIa
flanked by homology arms were validated by
T7 endonuclease assay and HDR-genotyping
and administered to haemophilia B mice.
The functional rescue was assessed
by coagulation function tests including
prothrombin time assay, FVIIa activity, and a
haemostatic challenge assay. The T7 assay
revealed a cleavage efficiency of 20–42%,
and further sequencing demonstrated the
targeted integration of mFVIIa into the
Rosa26 locus. The prothrombin time assay
on 8-week samples showed a significant
prothrombin reduction in mice that received
the gene-editing vectors (22%), and a
13% decline in mice that received only the
AAV-FVIIa in comparison to the mock-treated
animals; follow-up at 15 weeks showed a
similar finding. Coagulation FVIIa activity in
mice that received triple gene-editing vectors
was around 4-fold higher than in the mock
group. A tail-clip assay revealed a significant
reduction of blood loss in mice injected with
only FVIIa or gene editing vectors. Overall,
this study shows long-term expression and
phenotypic rescue with this novel geneediting
strategy. [OC 12.2]
Chen et al. looked at rescue of the
endogenous FVIII expression in haemophilia
A mice using CRISPR/Cas9 mRNA LNPs,
which provide an efficient gene delivery
system. LNPs carrying luciferase mRNA
were examined for transfection efficiency
in HepG2 and HUVEC cells. The efficiency
of two sgRNAs was examined in vitro using
a T7E1 assay. In vivo transfection efficiency
was investigated by immunofluorescent
staining of the liver following intravenous
injection of GFP mRNA LNPs into the mice.
Immunodeficient haemophilia A mice (NSG
HA) with indel mutation in FVIII exon 1 were
hydrodynamically injected with FVIII exon 1
targeting sgRNA/Cas9 expressing plasmid.
Subsequently, CRISPR/Cas9 mRNA LNPs
were synthesised and intravenously injected.
FVIII activity was examined by aPTT assay
and gene editing verified by DNA sequencing.
The results suggest that mRNA LNPs can
efficiently transfect both HepG2 and HUVEC
cells and can be delivered to hepatocytes
and LSECs in vivo. NSG HA mice treated
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ISTH 2022 Congress Review
with sgRNA/Cas9 plasmids regained FVIII
expression, and the subsequent CRISPR/
Cas9 mRNA LNPs successfully induced indel
mutation in mutant FVIII gene and rescued
endogenous FVIII activity. In combination,
these technologies have the potential to
repair FVIII mutations and recover FVIII gene
expression in patients with haemophilia A.
[OC 12.3]
Sternberg et al. investigated the safety and
efficacy of FVIII-QQ for AAV-mediated gene
therapy in CD4KO mice with haemophilia
A (HA/CD4KO). FVIII:C was determined
by CSA, and AAV-treated cohorts were
followed for survival or analysed by tailclip
assay. Recombinant hFVIII-WT or
hFVIII-QQ was intravenously administered
to immunocompetent haemophilia A mice
weekly for 7 weeks, and Bethesdatiter
determined. The vector dose EC50 and EC80
of blood loss post tail-clip for AAV-hFVIII-QQ
was 4.7- and 10-fold lower, respectively, than
Assay discrepancies
AAV-hFVIII-WT: supporting a 5- to 10-fold
benefit of the hFVIII-QQ transgene. Ongoing
studies of HA/CD4KO mice expressing
hFVIII-WT and hFVIII-QQ in the mild and
normal ranges of FVIII:C demonstrate no
significant survival differences for at least
3 months post-vector. Homozygous female
and hemizygous male mice endogenously
expressing FVIII-QQ via CRISPR-mediated
gene editing were viable, fertile, and the
same weight as their littermate controls.
Immunocompetent haemophilia A mice
treated with recombinant hFVIII-WT or
hFVIII-QQ demonstrated expected inhibitor
development that did not statistically differ.
The authors conclude that these data
demonstrate a haemostatic advantage
of using the FVIII-QQ transgene for AAVmediated
gene transfer. Preliminary murine
safety studies did not demonstrate evidence
of FVIII-QQ enhanced prothrombotic or
immunological risk relative to FVIII-WT.
[OC 12.4]
Transgene-derived FVIII activity varies depending on whether it is
measured with OSA or CSA. Understanding the mechanism of the
discrepancy is necessary to determine which assay best represents
in vivo haemostatic efficacy.
AAV-mediated gene transfer trials in
haemophilia A have demonstrated
transgene-derived FVIII:C is around 2-fold
higher by OSA than CSA; however, plasma
FVIII antigen concentrations correlate with
CSA-determined FVIII:C. The underlying
mechanism is unclear – particularly since
this is not observed with recombinant
proteins of the same amino acid sequence.
Possible hypotheses that may explain
the OSA- versus CSA-determined FVIII:C
discrepancy include enhanced activation, or
differences in the function of the transgenederived
FVIIIa species. Understanding the
reason for this discrepancy will determine
which assay best represents in vivo AAVderived
FVIII haemostatic efficacy, and
thereby patient management. Sternberg
et al. shared their efforts to elucidate the
mechanism behind AAV-derived FVIII assay
discrepancies. To probe these mechanisms,
AAV8-BDD-hFVIII was infused into male
HA/CD4KO or HA/vWF-/- mice, and FVIII:C
measured by OSA and CSA against a
standard curve of recombinant BDD-hFVIII
purified from BHK cells reconstituted in
mouse plasma. TGA and two-stage clotting
assays were also performed. As in humans,
Gene therapy in juveniles
the results showed that transgene-derived
FVIII:C determined by OSA was around 2-fold
higher than CSA-determined FVIII:C, and
FVIII:Ag closely correlated with CSA FVIII:C.
These data demonstrate that the discrepancy
is not species-specific to humans and is not
related to interactions with vWF. Interestingly,
FVIII:C by the two-stage assay was higher for
transgene-derived versus recombinant FVIII,
suggesting FVIIIa derived from gene therapy
has enhanced function relative to rFVIII.
The group intend to repeat these experiments
with human clinical samples to confirm
their findings. [OC 01.3]
The mass of the human liver doubles 8–9 months from birth, and again by
3–4 years. Liver growth and expanding blood volume may reduce Factor levels
in paediatric recipients of haemophilia gene therapy.
Nichols et al. determined the effect of
growth on FIX levels in male juvenile dogs
with haemophilia B following treatment
with an AAV-delivered, high-activity canine
FIX-R338L. The animals received an
AAV-encoding FIX-R338L variant under a liver
specific promoter at age 3 (n=6) or 6 months
(n=6) to model 2–6 or 6–12-year-old children,
respectively. Body weight, liver size, clinical
chemistries, and haemostatic activity were
monitored by WBCT, thromboelastography,
and aPTT. Dogs were followed for at least
12 months, during a period of liver growth
and blood volume expansion. Overall,
treatment was well tolerated. To date,
there have been no spontaneous bleeds
post-treatment. Increasing vector dose was
associated with progressive shortening of
clotting times. Stable, durable reductions
of WBCT, thromboelastography, and aPTT
values were observed in all dogs, indicative
of FIX activity. This durable efficacy was
observed in growing juvenile dogs treated
at an early age and was sustained for at
least 1 year despite liver growth and blood
volume expansion. [OC 21.4]
14 15

ISTH 2022 Congress Review
Bleeding tendency in haemophilia
Repeated bleeding in muscles and joints are major causes of morbidity and
disability in people with haemophilia. Novel therapies, including modified
replacement therapy and gene therapy, provide an opportunity to increase
FVIII activity, or to correct haemostasis. Information on bleeding phenotype in
haemophilia provides important information regarding the optimum target for
prophylactic treatment.
de Kovel et al. presented data from the
PedNet study group, assessing bleeding
according to baseline FVIII activity in 641
children with non-severe haemophilia A.
PedNet is a prospective, observational cohort
study across 32 HTCs. Those taking part had
baseline factor FVIII activity between 1–25%,
and were followed from diagnosis until either
01 January 2020, reaching 18 years, starting
prophylaxis, or inhibitor diagnosis. Onset of
bleeding and ABR were compared according
to categories of baseline FVIII. The median
age at first joint bleed was higher with
increasing FVIII activity, but overall ABR was
low, with 0.3 for all bleeds and 0.1 for joint
bleeds. The authors conclude that children
with non-severe haemophilia A have a low
bleeding tendency. Onset of joint bleeding
was delayed, and bleeding decreased with
increasing FVIII levels. Bleeding rates
approached zero at 16% for all bleeds, and
6% for joint bleeds. A steep decrease in
annual rates of joint bleeds was observed in
patients with FVIII >2%. [PB0181]
A post-hoc analysis of a non-interventional
study from Camp et al. compared bleeding
outcomes by prophylactic FVIII replacement
intensity. Study BMN 270-902 collected realworld
data on outcomes of a global cohort of
people with severe haemophilia A receiving
regular exogenous FVIII prophylaxis. The aim
was to verify consistency in bleeding outcomes
between those receiving high- and lowintensity
prophylaxis. Among 194 participants,
mean baseline annualised FVIII utilisation was
5499.6 and 2838.0 IU/kg/year for high- and
low-dose intensity cohorts, respectively, with
a mean annualised FVIII infusion rate of 146.7
and 120.2 infusions per year. Throughout
follow-up the absolute difference in mean
ABR for treated bleeds was 0.71 (P=0.4761),
and the absolute difference in the proportion
of participants with zero treated bleeds
was 0.7% (34.6% versus 35.3%; P=0.7708).
Similar trends were observed for all bleeds.
The authors concluded that the intensity of
prophylaxis did not impact bleeding outcomes
in study BMN 270-902, since bleeding risks
remain and were found to be similar across
the cohorts irrespective of FVIII consumption.
[PB0656]
Markson et al. further explored the topic
with a poster on medically recorded bleeds
and healthcare resource use in the United
States among men with haemophilia A.
Adult males aged 19–64 years prescribed
FVIII use during the on-study period
Mean (SD) IU/kg/year
8000
7000
6000
5000
4000
3000
2000
1000
0
Annualized FVIII utilization
5236.5
High-dose
prophylaxis
(n = 78)
P

ISTH 2022 Congress Review
Factor replacement and other therapies
New data were presented for recombination Factor, and non-replacement
products such as fitusiran.
Berger et al. showcased data from GENA-99 –
a prospective, multinational, non-interventional
study looking at the safety and efficacy of
simoctocog alfa in PTPs with haemophilia
A in routine clinical practice. Males with
haemophilia A (N=78) were enrolled in
13 countries and followed for 100 exposure
days. Over the course of the study, no patient
experienced an ADR, and no inhibitors were
reported. Of the 77 patients who received
prophylaxis, 41.6% had a 3-times per week
regimen, 23.4% were every-other-day,
and 22.1% took their prophylaxis twice per
week. In 74 patients who received at least
3 months of prophylaxis, mean duration was
10.2 months. Median ABR was comparable
to those in the clinical registration studies
(GENA-08 and GENA-03). In the 77 patients
who received prophylaxis, there were 246
breakthrough bleeding episodes; these were
treated with simoctocog alfa, with a success
rate of 88%. In the two patients treated ondemand,
there were 55 bleeding episodes,
98% of which were treated successfully with
simoctocog alfa. The authors argue that
these data indicate the long-term safety and
efficacy of simoctocog alfa in the real-life
setting are comparable to those in clinical
registration studies, and the drug is effective
and well-tolerated in patients of all ages in
routine clinical practice. [PB1126]
Fitusiran, an investigational siRNA
prophylactic, targets antithrombin mRNA
to rebalance haemostasis in people with
haemophilia A or B, irrespective of inhibitor
status. Srivastava et al. presented an analysis
of two Phase 3 studies to consumption with
fitusiran prophylaxis versus on-demand
BPA/CFC in people with haemophilia – with
or without inhibitors. Of 118 participants
randomised to fitusiran, 19 received
on-demand BPA, and 40 on-demand CFC.
Total consumption of aPCC and rFVIIa was
97.5% and 98.2% lower in the fitusiran versus
the on-demand BPA arm, and overall mean
consumption of FVIII and FIX was lower in the
fitusiran versus on-demand CFC arm: 95.9%
and 94.7%, respectively. The total number
of treated bleeds was lower with fitusiran
versus the on-demand BPA and CFC arms by
82.0% and 79.2%, respectively. This analysis
suggests that fitusiran prophylaxis reduced
total BPA/CFC consumption by ~95% or
more, thereby reducing treatment burden.
The reduction was driven by a decrease in
the total number of treated bleeds, injections
per bleed, and the BPA/CFC dose required.
[OC 40.3]
Pipe et al. also shared new data for fitusiran,
in their analysis of anti-thrombin levels and
thrombin generation from a Phase 3 study in
120 people with haemophilia A or B without
inhibitors. Participants were randomised 2:1
to receive once-monthly 80 mg subcutaneous
fitusiran prophylaxis, or on-demand factor
concentrates for 9 months. On Day 15, there
was a 71.6% mean reduction from baseline
in antithrombin levels in the fitusiran arm,
with a further reduction to 79.8% on Day 29,
and maintained at 85.3–88.6% from Day 43
onwards. There was a mean increase in TG
of 17.1 nM from baseline in the fitusiran arm
on Day 15, increasing to 24.4 nM on Day 29
and maintained at 29.8.1–43.1 nM from Day
43 onwards. These results corresponded
with an 89.9% reduction in estimated ABR
with fitusiran versus on-demand factor
concentrates. Fitusiran reached target
pharmacodynamic effect of antithrombin
lowering and increased thrombin generation
by Day 29 and demonstrated a consistent
effect throughout the study. The authors
conclude that these findings suggest fitusiran
has the potential to rebalance haemostasis
in people with haemophilia A or B without
inhibitors. [OC 50.2]
LIST OF ABBREVIATIONS
AAV – adeno-associated virus
ABR – annualised bleeding rate
ADR – adverse drug reaction
AE – adverse event
ALT – alanine aminotransferase
ApoE – apolipoprotein E
aPTT – activated partial
thromboplastin time
AST – aspartate transaminase
BDD – B-domain deleted
BHK – baby hamster kidney fibroblasts
BOEC – blood outgrowth endothelial cells
BPA – bypassing agent
CBC – complete blood count
CCL5 – chemokine ligand 5
CFC – clotting factor concentrate
CSA – chromogenic assay
EAHAD – European Association for
Haemophilia and Allied Disorders
EHC – European Haemophilia Consortium
EHCCC – European Haemophilia
Comprehensive Care Centre
EHTC – European Haemophilia
Treating Centre
FIX – Factor IX
FVIII – Factor VIII
GFP – green fluorescent protein
GTR – Gene Therapy Registry
hAAT – human alpha-one antitrypsin
HDR – homology directed repair
HRQoL – health-related quality of life
HSR – homologous recombination
HTC – haemophilia treatment centre
IL – interleukin
IRF7 – interferon regulatory factor 7
ISTH – International Society on
Thrombosis and Haemostasis
ITR – inverted terminal repeat
IV – intravenous
LNP – lipid nanoparticle
LSEC – liver sinusoidal endothelial cell
MDT – multidisciplinary team
MP – mannosylated peptide
Rangarajan et al. presented a similar
exploratory analysis in 60 participants
with inhibitors. On Day 15, there was a
76.8% mean reduction from baseline in
antithrombin activity levels in the fitusiran
arm, with a further reduction to 84.1% on
Day 29 and maintained at 87.6–89.9% from
Day 43 onwards. There was a mean increase
in peak thrombin generation of 21.9 nM
from baseline in the fitusiran arm on Day 15,
with a further increase to 30.8 nM on Day 29
and maintained at 36.8–47.5 nM from Day
43 onwards. These results translated into
a 90.8% reduction in estimated ABR with
fitusiran prophylaxis versus on-demand BPA.
These findings – and the significant reduction
in ABR – suggest fitusiran may rebalance
haemostasis and provide consistent bleed
protection in people with haemophilia A or B
with inhibitors. [PB1152]
NAb – neutralising antibody
NF-κB – nuclear factor kappa B
OSA – one-stage clotting assay
PBMC – peripheral blood
mononuclear cell
PTP – previously treated patient
PY – person-years
QoL – quality of life
RBC – red blood cell
RR – rate ratio
RWD – real-world data
TGA – thrombin generation assay
TLR – toll-like receptor
TNF – tumour necrosis factor
TTR – transthyretin
vg – vector genomes
vWF – von Willebrand factor
WBCT – whole blood clotting time
WFH – World Federation of Hemophilia
WT – wild type
18 19