Page 2273 - Hematology_ Basic Principles and Practice ( PDFDrive )

2020 Part XII Hemostasis and Thrombosis

for up to 4 weeks. In contrast, the approaches used to inhibit TFPI TABLE Features of Adeno-Associated Virus–Mediated Gene
have focused on functional neutralization using anti-TFPI aptamers, 135.10 Therapy
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antibodies and peptides. These approaches offer much promise but
are not without risks; a phase III study on an anti-TFPI aptamer was • AAV is a nonpathogenic virus.
halted because of unexpectedly increased bleeding with the product. • AAV rarely integrates into the host genome.
It remains unclear whether any of these rebalancing approaches to • There are no immediate adverse effects after AAV delivery.
hemostasis will provide sufficient procoagulant activity to eliminate • Innate immune reactivity to AAV is minimal.
bleeds in the absence of FVIII or FIX, or whether they would need • Vector readministration can be achieved with different vector
to be administered as adjunctive therapies with small doses of clotting serotypes (different capsids).
factor concentrate. Finally, the risk of generating a pathologic proco- • Cytotoxic T-cell responses to capsid protein presentation can limit
agulant response with these approaches remains a possibility that will the duration of expression.
require careful monitoring. • Transgene size is limited to ~5 kb.
The other innovative approach is the use of a bispecific antibody AAV, Adeno-associated virus.
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that substitutes for the cofactor activity of FVIIIa. This antibody
binds to FIXa and FX and at least partially mimics the scaffold
function of FVIIIa in the intrinsic tenase complex. The antibody can
be administered by weekly subcutaneous injection and phase II clini- transfer with these vectors is limited to tissues in which a significant
cal studies have shown no adverse effects and a reduction in bleeding proportion of cells are cycling. In contrast, lentiviral vectors are
events in hemophilia A patients both with and without FVIII inhibi- equally capable of transducing both postmitotic and replicating cells.
tors. Therefore the bispecific antibody could serve as an alternative For this reason, most of the more recent studies of retroviral hemo-
to FVIII replacement in hemophilia A patients with or without philia gene transfer have used lentiviral vector protocols in which the
inhibitors. vector construct is usually derived from elements of the human
immunodeficiency virus. The other major difference between lenti-
viral gene delivery and gene transfer with other viral vectors is that
Gene Therapy for Hemophilia lentiviruses integrate their genome into the recipient cell genome as
a natural part of their life cycle. Although viral vectors do not possess
Since the cloning of the FVIII and FIX genes in the early 1980s, the structural components that enable further rounds of viral replica-
hemophilia has been a leading candidate for the application of tion, there is a risk of insertional mutagenesis, which can trigger
somatic cell gene transfer. The rationale for gene therapy in hemo- activation of adjacent oncogenes or inactivation of tumor suppressor
philia includes the following: small increments in FVIII or FIX levels loci. Studies performed in the past 5 years indicate that lentiviral
have a significant clinical benefit; regulation of FVIII or FIX levels is integrations are not random but tend to cluster in transcribed regions
not critical as long as they do not reach supraphysiologic levels for of the genome and more often occur within introns and coding
extended periods of time; the site of coagulation transgene expression regions of genes rather than in the upstream regulatory regions where
need not be restricted as long as sufficient amounts of the protein γ-retroviruses insert.
reach the circulation; and small (genetically modified hemophilic Currently, the lead candidate for hemophilia gene transfer is AAV
mice) and large (spontaneously generated hemophilic dogs) animal (Table 135.10). This is a small nonpathogenic human parvovirus
models of hemophilia are available for preclinical evaluation of gene with a small, single-stranded DNA genome of approximately 4.8 kb.
transfer approaches. Infection in humans, which for some serotypes of the virus is frequent,
is not associated with any clinical disease. The various serotypes of
AAV have distinct tissue tropisms, and thus by using the capsid
Modes of Transgene Delivery sequence for a particular serotype, gene therapists can target specific
tissues for transgene expression. As one example, AAV-8 has excellent
A key component to any gene transfer strategy is the development of hepatotropic properties and has been successfully used for liver
a delivery system that enables efficient transfer of the therapeutic transgene delivery and expression. Upon entry into recipient cells,
transgene to the recipient cell type of choice. After 30 years of AAV is maintained within the nucleus in the form of stable extra-
investigation, viral vectors remain the most effective means of achiev- chromosomal concatemers, and only a small proportion of the virus
ing high-level gene transfer. Although efforts have been made to integrates into the recipient genome at sites of natural chromosomal
enhance the efficiency of nonviral delivery methods such as liposome breaks. Wild-type AAV preferentially integrates into a site on chro-
encapsulation, various physicochemical conjugates, and hydrody- mosome 19, but the replication-defective AAV vectors appear to
namic injection, all of these approaches have limitations that preclude insert into sites of natural chromosomal breakages. One significant
their advancement into the clinic at this time. limitation to AAV vectors is their small packaging capacity. Transgenes
Hemophilia gene transfer studies have used three main types of larger than 5 kb limit the capacity for viral particle assembly, and
viral vector: adenovirus, several forms of retrovirus and adeno- although this is not a problem for FIX (cDNA ≈1.4 kb), even the B
associated virus (AAV). Trials of adenoviral gene transfer have been domain deleted forms of FVIII (cDNA of ≈4.7 kb) are not easily
successful in animal models of hemophilia, but the single hemophilia accommodated by this vector type.
A patient treated with an adenoviral vector experienced significant
hematologic toxicity, and no further clinical studies have been under-
taken with this vector type. Thus although adenoviral gene transfer Clinical Trials of Hemophilia Gene Therapy
is highly efficient, these vectors elicit a major innate immune response
upon cell entry, and the proinflammatory consequences of this Approximately 100 patients with hemophilia have undergone clinical
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response remain a significant safety concern. Therefore until the evaluation of gene transfer strategies. An initial trial of ex vivo gene
innate immune reactivity of adenoviral gene delivery has been miti- transfer for FIX was performed on two subjects in China in the
gated, this vector system will not be used for the treatment of 1990s. A FIX γ-retroviral vector was used to transduce autologous
hemophilia. skin-derived fibroblasts before reimplantation. No significant increases
The second viral vector approach that has shown promise in in FIX levels were observed. Since then, one patient has been treated
hemophilia is the retroviral system. Initial studies were performed with an intravenous FVIII adenoviral vector; a plasmid vector was
with replication-defective γ-retroviral vectors, and one human clinical used as a second ex vivo approach in which autologous fibroblasts
study in hemophilia A also used this vector system. The major were implanted into the omentum, and a small cohort of patients
problem with these vectors is the requirement for recipient cell rep- with hemophilia was treated with intravenous FVIII γ-retrovirus.
lication to facilitate nuclear entry of the vector. Consequently, gene Aside from the transient thrombocytopenia associated with adenoviral