June 20, 2021

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Explore experimental AAV gene therapy for patients with hemophilia

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Explore experimental AAV gene therapy for patients with hemophilia



Explore experimental AAV gene therapy for patients with hemophilia.  Gene therapy has the potential to overcome many limitations in preventive hemophilia treatment.

Some clinical trials have evaluated adeno-associated virus (AAV)-mediated treatment of hemophilia type A and type B research gene transfer methods, and the practical application of these methods Subtle differences are due to differences in AAV serotypes, genetic modification, manufacturing, administration, administration and follow-up methods.

The basic steps of gene therapy are similar to the process of preparing and delivering packages (Figure 1). The delivery package is a viral vector, the gene is the item in the package-a functional copy of F8 or F9-and the liver is the delivery address. Table 1 provides Glossary of key gene therapy terms.

Explore experimental AAV gene therapy for patients with hemophilia

Figure 1 The AAV-mediated gene transfer process is similar to the preparation and delivery of packaging

Table 1 Glossary of important terms
Explore experimental AAV gene therapy for patients with hemophilia

Wild-type AAVs (wtAAV) are members of the Parvoviridae and Parvovirus-dependent genus. They are not pathogenic in humans and have replication defects (lack of the ability to reproduce independently).

wtAAV is a single-stranded DNA virus. The genome consists of two open reading frames called rep and cap, flanked by terminal inverted repeats; Rep encodes the protein responsible for replication, and cap encodes the structural protein that constitutes the viral capsid . Recombinant AAV (rAAV) DNA lacks replication in rep and cap (Figure 2).

In the absence of helper viruses and viral replication genes, rAAVDNA is randomly integrated into the host genome at a low frequency (≤0.01% in one study), reducing the concern about the genotoxicity of other vectors, such as retroviruses and lentiviruses .

Explore experimental AAV gene therapy for patients with hemophilia

Figure 2 Transgene expression using liver-targeted AAV vector


The package contents and transportation methods are as follows: rAAV vector is produced by replacing the rep and cap genes with single-stranded transgene expression cassettes that contain therapy-related genes and other non-coding transcriptional regulatory elements, such as promoting transgenes in certain cell types The expressed promoter sequence, the expression cassette is inserted between the inverted repeats at the end of the palindrome (Figure 2).

The purified vector is transported to the local center and cryopreserved, and then thawed by the pharmacist and prepared for infusion. It is given a single intravenous outpatient infusion according to body weight. The capsid binds to the target cell and is internalized through receptor-mediated endocytosis. , Release the transgene, and then degrade.

Since the rep sequence (which promotes the integration of the wtAAV genome) is not present in the final vector, the transgene forms an end-to-end exome concatenation containing multiple repetitive DNA genomes, but usually does not integrate into the host genome.

Even chromosomes allow continuous transgene expression because they are stable for a long time in non-dividing cells and will not cause damage to target cells. Single-stranded AAV vectors need to synthesize or recruit complementary (c) DNA strands (or self-annealing of complementary sequences in caav vectors) to provide a double-stranded DNA template as an RNA transcription start site. Then transcription and translation are carried out to produce a functional coagulation factor protein (Figure 2).

As of this writing, the health department has not approved hemophilia gene therapy products for routine clinical use, but patient selection criteria may soon become relevant considerations and may be based on the selection criteria of clinical trials (Figure 3).

Explore experimental AAV gene therapy for patients with hemophilia

Figure 3 Selection criteria for potential participants in clinical trials of gene therapy for hemophilia

So far, patients participating in clinical trials are mainly limited to adult male patients with baseline endogenous coagulation factor activity ≤1-2%; other challenges posed by extending gene therapy to the pediatric population may further delay the development of this important patient group Treatment, the durability of the effect in pediatric patients may be limited.

After the gene therapy research product is injected, if the gene transfer is successful, the use of exogenous factors usually decreases significantly due to the increased expression of endogenous factors; if the gene therapy is unsuccessful or the durability is limited, research participants may return To other prevention options, such as factor replacement therapy or imiizumab.

Trial participants must commit to frequent intensive follow-up of liver transaminase assessment in the first few months after fusion. The increase in liver enzymes occurs before or at the same time as transgene expression and potency loss, usually in the first 12 weeks after vector infusion. , Supportive intervention after the detection of an increase in transaminase may help prevent the loss of transgene expression.

Compared with the preventive use of corticosteroids to prevent loss of transgene expression, further studies are needed to determine the effectiveness of immunomodulation methods in maintaining transgene expression in patients with vector-related liver inflammation.

The coreHEM project has established a set of results to evaluate the effectiveness, safety, and value of gene therapy for hemophilia (Figure 4); these results provide a useful focus when discussing patient expectations before treatment, and patients should be informed about treatment The results that will be monitored during and after treatment are different from the follow-up of factor replacement therapy they have previously experienced.

Figure 4 The main results that need to be monitored after receiving research gene therapy

The CoreHEM project has determined the core efficacy results of six gene therapy trials, divided into several independent areas: physiology, clinical (frequency of bleeding events, factor activity level and expression time); pain, discomfort (chronic pain); resource use ( Direct cost of the use of the healthcare system) and mental health (emotional function).

When discussing expectations with potential gene therapy subjects, it should be clear that mental and physical health will be monitored throughout the treatment process.

The coreHEM project also identified 8 safety results, divided into 3 health areas, and recommended for gene therapy clinical trial design:

(1) Short-term AEs (liver meridian amiitis, short-term immune response to FVIII/FIX [inhibitor development] and immune response to gene therapy [humoral and cell-mediated immunity; memory, cytotoxicity and potential innate immunity] , Thrombosis);

(2) Long-term AEs (occurrence of other diseases, vector integration into host genome, vector neutralization and duration of response)

(3) Mortality (cause of death).


The gene therapy methods discussed in this review target somatic cells instead of germ cells. Therefore, gene correction will not be passed on to children. Patients should be made aware that there may be vectors in their body fluids after treatment, and patients should be advised to treat them. Use barrier contraception afterwards to reduce any possible risk of horizontal transmission.

The practical points of this article are:

  • The experimental AAV-mediated liver-directed gene therapy process can be described by the “package delivery” analogy, where the carrier is the package to be delivered and the liver is the delivery address;
  • The actual application of AAV-mediated gene therapy may vary according to the selection of AAV serotypes, genetic modification, manufacturing, drug delivery, drug delivery and long-term follow-up methods;
  • Whether patients with hemophilia are suitable to participate in research trials of gene therapy depends on many factors, including gender, age, disease severity, the presence of factor inhibitors, AAV neutralizing antibodies, and comorbidities;
  • The key efficacy and safety results of hemophilia gene therapy trials provide a useful focus when managing patient expectations for gene therapy;
  • Effective discussions on gene therapy for hemophilia are of vital importance to patients and healthcare providers, especially in the application of permission for gene therapy for hemophilia treatment is expected in 2020.



It is hoped that the information provided here can promote information and collaborative discussions between healthcare providers and potential participants in clinical trials of gene therapy for hemophilia research.


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