Gene therapy for hemophilia A achieves long-term curative effect

Hemophilia is an invisible X-linked hereditary hemorrhagic disease affecting males, including hemophilia A and B
.

In the male population, the incidence of hemophilia A is about 1 in 5,000, while the incidence of hemophilia B is about 1/25,000
.

At present, lifelong exogenous coagulation factor replacement therapy is still the main treatment for hemophilia
.

Because hemophilia has the characteristics of single-gene inheritance and a wide therapeutic window for coagulation factors, it has become one of the first choices for gene therapy
.

In recent years, great progress has been made in the field of gene therapy for hemophilia at home and abroad.
The New England Journal of Medicine (NEJM) has published a number of research results on gene therapy for hemophilia.
These studies show that gene therapy has significantly improved hemophilia The patient's condition, but there are also certain shortcomings
.

On November 28, 2021, NEJM re-published the long-term follow-up results of the Phase 1/2 trial of gene therapy for hemophilia A.
The results showed that of the 18 patients treated, 16 patients did not require prophylactic infusion of coagulation factors and were bleeding Incidents have been drastically reduced
.

"NEJM Frontiers in Medicine" specially invited Professor Zhang Lei from the Institute of Hematology, Hospital of Hematology, Chinese Academy of Medical Sciences, to interpret the trial and comprehensively comment on the progress of gene therapy for hemophilia at home and abroad
.

Professor Zhang Lei has been researching gene therapy for many years and has taken the lead in conducting clinical trials of gene therapy for hemophilia in China
.

To read the full text translation, please visit the "NEJM Medical Frontiers" official website, APP or click on the picture of the WeChat applet
.

Zhang Lei, Institute of Hematology, Hospital of Hematology, Chinese Academy of Medical Sciences; National Clinical Research Center for Hematological Diseases.
Hemophilia is an X-linked congenital bleeding disorder, which is caused by F8 or F9 single gene mutations leading to clotting factor Ⅷ (hemophilia A) Or it is caused by insufficient secretion of blood coagulation factor IX (hemophilia B) or functional defects.
It is mainly manifested by repeated bleeding in joints and muscles.
It often starts at a young age and involves lifelong
.

Most patients with severe hemophilia need blood clotting factor replacement therapy for life, and receive intravenous injections of clotting factors multiple times a week to prevent spontaneous bleeding.
If preventive treatment is not standardized, patients often suffer from repeated joint bleeding and cause varying degrees of limbs.
Dysfunction and even disability, severe bleeding can even lead to death
.

Hemophilia is a single-gene genetic disease, and the FⅧ or FⅨ activity level rising to 5% of the normal value can significantly improve the bleeding symptoms of patients
.

Gene therapy as an emerging therapy has great advantages for hemophilia.
The long-term stable expression of coagulation factor genes in patients can be obtained through a one-time intravenous administration, and gene expression can be evaluated by detecting the level of coagulation factors in plasma
.

Therefore, the success of gene therapy will bring huge clinical benefits to patients, greatly improve the quality of life, and at the same time reduce the long-term heavy medical burden of patients and society
.

AAV gene therapy for hemophilia A achieves long-term efficacy November 28, 2021, the New England Journal of Medicine (NEJM) published the long-term follow-up results of the Phase 1/2 clinical trial of hemophilia A gene therapy, 18 patients participated in the treatment In 16 patients, good treatment results were achieved, and bleeding symptoms were significantly reduced.
At the same time, studies believe that the treatment has good safety [1]
.

In this study, 18 patients with severe hemophilia A received gene therapy targeting the liver with adeno-associated virus (AAV) as a vector
.

The median follow-up time was 36.
6 months, of which 2 patients lost expression of Ⅷ due to the anti-AAV immune response, and the other 16 patients maintained FⅧ expression at a certain level, and 12 of them were followed up for more than 2 years
.

The average FⅧ expression level was 12.
9±6.
9% in patients who were followed up for 26 weeks to 52 weeks, and the FⅧ expression level was 12.
0±7.
1% in patients who were followed up for more than 52 weeks
.

Compared with before gene therapy, the annualized bleeding rate after treatment decreased by 91.
5%
.

In terms of safety, 8 patients had 33 treatment-related side effects, 17 of which were related to AAV vector infusion, mainly including fever, muscle pain, and vomiting, and 7 patients had elevated liver transaminases, which improved after treatment
.

After more than 50 months of follow-up, the researchers believe that AAV gene therapy has good safety
.

Research on gene therapy for hemophilia at home and abroad.
International gene therapy for hemophilia based on AAV vector started 20 years ago.
Researchers injected AAV-FⅨ into the muscles of patients with hemophilia B.
The process is safe.
Muscle biopsy shows FⅨ expression.
It lasts for more than 3 years, but the plasma FⅨ level is very low
.

At the same time, preclinical studies have shown that AAV vector delivered to the liver by vascular injection can achieve a higher level of FⅨ expression than intramuscular injection
.

In 2006, the first clinical trial of AAV2 vector gene therapy was conducted in patients with severe hemophilia B.
The vector was infused through the hepatic artery, but the immune response caused FIX expression to last only about 2 months [2]
.

In 2011, Nathwani et al.
gave the liver-targeted AAV8 vector through intravenous infusion for the first time in patients with hemophilia B, and the continuous expression level of FⅨ was 1% to 6%.
The bleeding events of patients were significantly reduced and there was no need for preventive use of FⅨ [3]
.

After 3 years, the median FⅨ level of 6 patients in the highest dose group was about 5%.
Some patients have been followed up for more than 10 years, and the F9 gene can be expressed stably for a long time [4]
.

In December 2017, George et al.
published the results of a phase 1/2 clinical trial.
The AAV vector carries a liver-specific promoter and a codon-optimized highly active single point mutation F9 (Padua)
.

F9-Padua is a naturally occurring missense mutant located in the FIX amino acid sequence R338L in humans, which can increase the activity of FIX by 7 to 8 times.
The steady-state FⅨ activity of 10 patients with hemophilia B who received this treatment was 14% to 81% of the normal value, long-term follow-up has achieved good results [5]
.

Due to the limited packaging capacity of AAV (wild-type AAV is 4680 nucleotides), the application of this vector in hemophilia A gene therapy is limited.
Rangarajan et al.
used the FⅧ coding sequence (4.
3 kb) deleted from the B-domain.
) Reduced the size of the F8 gene expression cassette
.

Using this method, researchers used the codon-optimized AAV5-hFⅧ-SQ vector to conduct a phase 1/2 clinical trial in patients with severe hemophilia A.
In the high-dose group (6×1013 vg/kg), 6 out of 7 patients In 1 year, FⅧ level was >50% [6].
After three years of follow-up, the average FⅧ level of patients dropped to 20%, but the bleeding events of these patients were significantly reduced [7]
.

China reported the world's first clinical trial of gene therapy for hemophilia B in 1996 [8], using retroviral vectors to transfect human F9 gene into patient's isolated skin fibroblasts, and inject it into the patient's abdominal or back subcutaneous tissue
.

The plasma FⅨ level in the two patients increased more than twice and lasted for more than 1 year
.

In November 2019, our team and Professor Xiao Xiao's team from East China University of Science and Technology launched China's first AAV-based liver-targeted hemophilia B gene therapy clinical trial (NCT04135300)
.

The study has completed the enrollment of all 10 subjects, all receiving AAV-FⅨ-R338L-Padua treatment (5×1012 vg/kg, single intravenous injection).
The first patient was followed up for more than 2 years after treatment, with a median follow-up The time reached 42 weeks, the median FⅨ activity reached 36.
75%, and the median number of patients with annual bleeding was 0, which was consistent with the effectiveness and safety of international reports (unpublished)
.

The AAV gene therapy product has been approved by the National Medical Products Administration for New Drug Clinical Trial (IND) in August 2021, to carry out gene therapy clinical trials for adult male hemophilia B patients
.

In 2021, our team will carry out the recruitment of the hemophilia A gene therapy clinical trial (NCT04728841).
At present, 3 patients have been enrolled.
The follow-up for 26 weeks showed that the level of factor Ⅷ of the patients was significantly increased by an average of 20%.
After the gene therapy, the patients There were no bleeding incidents
.

Problems to be solved AAV is a type of single-stranded DNA-deficient virus with a simple structure.
Its replication and packaging require the cooperation of a helper virus (usually an adenovirus or herpes virus).
Many years of research believe that AAV will not cause any human diseases
.

It has a variety of natural serotypes and engineered variants that can infect almost all types of tissues, including non-dividing cells
.

Unlike adenovirus, AAV rarely causes a strong immune response.
It has the characteristics of high safety, wide host application range and long expression time in the body, making it the most promising gene therapy vector for hemophilia
.

However, AAV gene transduction still has potential risks, including immunogenicity, hepatotoxicity, occasional insertion mutation, gene-specific toxicity, and limited durability [9]
.

First, liver toxicity is the most common adverse reaction observed in the clinic
.

One of the first problems in the early stage of AAV gene therapy is the host's immune response to the hepatocytes transduced by the virus
.

Our research shows that short-term use of steroid hormones such as prednisone can control the increase in serum transaminases, and no evidence of later recurrence or persistent liver cell damage has been observed.
The process of lowering liver enzymes seems to be a self-limiting phenomenon.
A study also showed similar side effects
.

Therefore, how to reduce the immune response and liver cell damage caused by AAV should be actively sought
.

Second, another potential risk of AAV vector liver gene therapy is genotoxicity caused by possible insertion mutations
.

In a dog model followed up for 10 years in the AAV gene therapy trial of hemophilia A, it has been confirmed that the clonal expansion of hepatocytes is related to it, but no evidence of canceration was found [10]
.

In addition, an adult hemophilia B patient with chronic hepatitis B and C infections for many years was treated with intravenous AAV5-hFⅨ Padua.
Hepatocellular carcinoma was found during the 1-year follow-up, but the integration of AAV vectors was not detected in the tumor cells
.

It has also been reported that there is integration of small fragments of wild-type AAV2 DNA in the genome of a small number of human liver cancer samples
.

Therefore, for high-risk groups of hemophilia patients receiving gene therapy, such as patients with chronic hepatitis and liver fibrosis, genetic sequencing of their liver tissues will help assess the integration probability of AAV and the risk of liver cancer
.

Third, the variability and persistence of FⅧ gene expression are related to vector dosage, vector serotype, promoter tissue specificity, transgene efficiency and host differences
.

The variability of FⅧ gene expression and the potential toxicity with the increase of vector dose are still issues that need to be considered
.

Some FⅧ gene subjects decreased FⅧ expression by more than 50% after 5 years, indicating that the persistence of gene expression in AAV hemophilia gene therapy is still a challenge [1,8]
.

Fourth, about 20% to 70% of hemophilia patients have pre-existing neutralizing antibodies against specific AAV serotypes, and the seropositivity rate increases with age and exposure to plasma products
.

Preclinical studies have proved that humoral immunity can prevent the successful transduction of AAV into liver cells, which will hinder the effective gene transfer of AAV
.

At present, patients with pre-existing antibodies have been excluded from gene therapy trials, but the existence of neutralizing antibodies is the main limitation of gene therapy for hemophilia patients
.

Methods such as immunosuppressive agents, plasma exchange, increasing carrier doses or increasing empty capsids may reduce the obstacles to neutralizing antibodies
.

Future prospects At present, a number of studies on gene therapy of hemophilia with AAV as a carrier are in the phase 2/3 phase of clinical trials, and it is expected that hemophilia gene therapy products may be approved in the next 1-2 years
.

However, how to obtain the best efficacy and safety of gene therapy for hemophilia is the focus of current scientists.
Future research directions will focus on the design of AAV gene vectors to minimize immunogenicity, reduce host immune response and Avoid target organ toxicity and genotoxicity
.

With the continuous development of gene therapy research, new gene editing technologies continue to emerge.
For example, the CRISPR-Cas9 gene editing method is used to repair specific gene mutation sites in hemophilia to achieve the purpose of individualized gene therapy for hemophilia
.

Therefore, gene therapy for hemophilia in the future is expected to achieve safe, efficient and long-lasting expression of coagulation factors, and become an effective way to cure hemophilia patients
.

References 1.
George LA, Monahan PE, Eyster E, et al.
Multiyear factor Ⅷ expression after AAV gene transfer for hemophilia A.
N Engl J Med 2021;385:1961-73.
2.
Manno CS, Pierce GF, Arruda VR, et al.
Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.
Nat Med 2006;12:342-7.
3.
Nathwani AC, Tuddenham EG, Rangarajan S, et al.
.
Adenovirus-associated virus vector-mediated gene transfer in hemophilia B.
N Engl J Med 2011;365:2357-65.
4.
Nathwani AC, Reiss UM, Tuddenham EG, et al.
.
Long-term safety and efficacy of factor Ⅸ gene therapy in hemophilia B.
N Engl J Med 2014;371:1994-2004.
5.
George LA, Sullivan SK, Giermasz A, et al.
.
Hemophilia B gene therapy with a high-specific-activity factor Ⅸ variant.
N Engl J Med 2017;377: 2215-27.
6.
Rangarajan S, Walsh L, Lester W, et al.
.
AAV5-factor Ⅷ gene transfer in severe hemophilia A.
N Engl J Med 2017;377:2519-30.
7.
Pasi KJ, Rangarajan S, Mitchell N, et al.
.
Multiyear follow-up of AAV5-hFⅧ-SQ gene therapy for hemophilia A.
N Engl J Med 2020;38:29-40.
8.
X Qiu, D Lu, J Zhou, et al.
Implantation of autologous skin fibroblast genetically modified to secrete clotting factor Ⅸ partially corrects the hemorrhagic tendencies in two hemophilia B patients.
Chin Med J (Engl) 1996;109:832-9.
9.
Ragni MV.
Hemophilia as a blueprint for gene therapy.
Science 2021;374:40-41.
10.
Donsante A, Miller DG, Li Y, et al.
AAV vector integration sites in mouse hepatocellular carcinoma.
Science 2007;317:477.
Author introduction Zhang Lei, Doctor of Medicine, Chief Physician, Doctoral Supervisor, Distinguished Professor of "Concord Scholar"N Engl J Med 2020;38:29-40.
8.
X Qiu, D Lu, J Zhou, et al.
Implantation of autologous skin fibroblast genetically modified to secrete clotting factor Ⅸ partially corrects the hemorrhagic tendencies in two hemophilia B patients.
Chin Med J (Engl) 1996;109:832-9.
9.
Ragni MV.
Hemophilia as a blueprint for gene therapy.
Science 2021;374:40-41.
10.
Donsante A, Miller DG, Li Y, et al.
AAV vector integration sites in mouse hepatocellular carcinoma.
Science 2007;317:477.
Author introduction Zhang Lei, Doctor of Medicine, Chief Physician, Doctoral Supervisor, Distinguished Professor of "Concord Scholar"N Engl J Med 2020;38:29-40.
8.
X Qiu, D Lu, J Zhou, et al.
Implantation of autologous skin fibroblast genetically modified to secrete clotting factor Ⅸ partially corrects the hemorrhagic tendencies in two hemophilia B patients.
Chin Med J (Engl) 1996;109:832-9.
9.
Ragni MV.
Hemophilia as a blueprint for gene therapy.
Science 2021;374:40-41.
10.
Donsante A, Miller DG, Li Y, et al.
AAV vector integration sites in mouse hepatocellular carcinoma.
Science 2007;317:477.
Author introduction Zhang Lei, Doctor of Medicine, Chief Physician, Doctoral Supervisor, Distinguished Professor of "Concord Scholar"Miller DG, Li Y, et al.
AAV vector integration sites in mouse hepatocellular carcinoma.
Science 2007;317:477.
Author introduction Zhang Lei, Doctor of Medicine, Chief Physician, Doctoral Supervisor, Distinguished Professor of "Concord Scholar"Miller DG, Li Y, et al.
AAV vector integration sites in mouse hepatocellular carcinoma.
Science 2007;317:477.
Author introduction Zhang Lei, Doctor of Medicine, Chief Physician, Doctoral Supervisor, Distinguished Professor of "Concord Scholar"
.

Deputy Dean of the Institute of Hematology, Hospital of Hematology, Chinese Academy of Medical Sciences, Deputy Director of the National Clinical Research Center for Hematological Diseases, Deputy Director of the State Key Laboratory of Experimental Hematology, Director of the Key Laboratory of Gene Therapy, Chinese Academy of Medical Sciences, Tianjin Hematology Gene Director of the Key Laboratory of Treatment, Director of Tianjin Blood and Regenerative Medicine
.

Engaged in the diagnosis and treatment of blood coagulation diseases and platelet diseases, published more than 100 SCI papers in professional journals at home and abroad such as Cell Stem Cell, Blood and Leukemia, applied for 4 patents, won 1 national award, and won provincial and ministerial awards 4 Item
.

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.

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