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Feds aim to streamline gene therapy oversight
“As the NIH, the FDA, and research entities have moved to strengthen their individual oversight efforts, some overlaps have occurred,” Dr. Collins and Dr. Gottlieb wrote Aug. 15 in the New England Journal of Medicine. “Substantial duplication has arisen in the submission of initial protocols, annual reports, amendments, and reports of serious adverse events. Originally, these overlaps – which affect no other field in biomedical research – were viewed as harmonized reporting that enabled FDA to conduct regulatory oversight while maintaining confidentiality with sponsors and allowed NIH to provide transparency with regard to the research.”
With three approved treatments – two CAR T therapies and a treatment for a genetic retinal dystrophy – and more than 700 active investigational new drug applications in front of the FDA, “it seems reasonable to envision a day when gene therapy will be a mainstay of treatment for many diseases,” according to the editorial.
To remedy the situation, NIH officials Aug. 16 posted a proposed update to NIH guidance that seeks “to reduce the duplicative oversight burden by further limiting the role of NIH and RAC [Recombinant DNA Advisory Committee] in assessing gene therapy protocols and reviewing their safety information,” Dr. Collins and Dr. Gottlieb wrote. “Specifically, these proposals will eliminate RAC review and reporting requirements to the NIH for human gene therapy protocols. They will also revise the responsibility of institutional Biosafety Committees, which have local oversight for this research, making their review of human gene therapy protocols consistent with review of other research subject to the NIH Guidelines. Such streamlining will also appropriately place the focus of the NIH Guidelines squarely back on laboratory biosafety.”
RAC was originally established in 1974 to advise the NIH director on research involving manipulation of nucleic acids, but was later expanded to review and discuss protocols for gene therapy in humans.
The proposed structure provides an opportunity “to return the RAC to the spirit in which it was founded. ... The NIH envisions using the RAC as an advisory board on today’s emerging technologies, such as gene editing, synthetic biology, and neurotechnology, while harnessing the attributes that have long ensured its transparency,” they wrote.
This latest proposal follows a suite of FDA draft guidances on gene therapies issued in July that proposed new guidance on manufacturing issues, long-term follow-up, and pathways for clinical development in certain areas.
The guidance is scheduled for publication in the Federal Register on Aug. 17.
SOURCE: Collins FS and Gottlieb S. N Engl J Med. doi: 10.1056/NEJMp1810628.
“As the NIH, the FDA, and research entities have moved to strengthen their individual oversight efforts, some overlaps have occurred,” Dr. Collins and Dr. Gottlieb wrote Aug. 15 in the New England Journal of Medicine. “Substantial duplication has arisen in the submission of initial protocols, annual reports, amendments, and reports of serious adverse events. Originally, these overlaps – which affect no other field in biomedical research – were viewed as harmonized reporting that enabled FDA to conduct regulatory oversight while maintaining confidentiality with sponsors and allowed NIH to provide transparency with regard to the research.”
With three approved treatments – two CAR T therapies and a treatment for a genetic retinal dystrophy – and more than 700 active investigational new drug applications in front of the FDA, “it seems reasonable to envision a day when gene therapy will be a mainstay of treatment for many diseases,” according to the editorial.
To remedy the situation, NIH officials Aug. 16 posted a proposed update to NIH guidance that seeks “to reduce the duplicative oversight burden by further limiting the role of NIH and RAC [Recombinant DNA Advisory Committee] in assessing gene therapy protocols and reviewing their safety information,” Dr. Collins and Dr. Gottlieb wrote. “Specifically, these proposals will eliminate RAC review and reporting requirements to the NIH for human gene therapy protocols. They will also revise the responsibility of institutional Biosafety Committees, which have local oversight for this research, making their review of human gene therapy protocols consistent with review of other research subject to the NIH Guidelines. Such streamlining will also appropriately place the focus of the NIH Guidelines squarely back on laboratory biosafety.”
RAC was originally established in 1974 to advise the NIH director on research involving manipulation of nucleic acids, but was later expanded to review and discuss protocols for gene therapy in humans.
The proposed structure provides an opportunity “to return the RAC to the spirit in which it was founded. ... The NIH envisions using the RAC as an advisory board on today’s emerging technologies, such as gene editing, synthetic biology, and neurotechnology, while harnessing the attributes that have long ensured its transparency,” they wrote.
This latest proposal follows a suite of FDA draft guidances on gene therapies issued in July that proposed new guidance on manufacturing issues, long-term follow-up, and pathways for clinical development in certain areas.
The guidance is scheduled for publication in the Federal Register on Aug. 17.
SOURCE: Collins FS and Gottlieb S. N Engl J Med. doi: 10.1056/NEJMp1810628.
“As the NIH, the FDA, and research entities have moved to strengthen their individual oversight efforts, some overlaps have occurred,” Dr. Collins and Dr. Gottlieb wrote Aug. 15 in the New England Journal of Medicine. “Substantial duplication has arisen in the submission of initial protocols, annual reports, amendments, and reports of serious adverse events. Originally, these overlaps – which affect no other field in biomedical research – were viewed as harmonized reporting that enabled FDA to conduct regulatory oversight while maintaining confidentiality with sponsors and allowed NIH to provide transparency with regard to the research.”
With three approved treatments – two CAR T therapies and a treatment for a genetic retinal dystrophy – and more than 700 active investigational new drug applications in front of the FDA, “it seems reasonable to envision a day when gene therapy will be a mainstay of treatment for many diseases,” according to the editorial.
To remedy the situation, NIH officials Aug. 16 posted a proposed update to NIH guidance that seeks “to reduce the duplicative oversight burden by further limiting the role of NIH and RAC [Recombinant DNA Advisory Committee] in assessing gene therapy protocols and reviewing their safety information,” Dr. Collins and Dr. Gottlieb wrote. “Specifically, these proposals will eliminate RAC review and reporting requirements to the NIH for human gene therapy protocols. They will also revise the responsibility of institutional Biosafety Committees, which have local oversight for this research, making their review of human gene therapy protocols consistent with review of other research subject to the NIH Guidelines. Such streamlining will also appropriately place the focus of the NIH Guidelines squarely back on laboratory biosafety.”
RAC was originally established in 1974 to advise the NIH director on research involving manipulation of nucleic acids, but was later expanded to review and discuss protocols for gene therapy in humans.
The proposed structure provides an opportunity “to return the RAC to the spirit in which it was founded. ... The NIH envisions using the RAC as an advisory board on today’s emerging technologies, such as gene editing, synthetic biology, and neurotechnology, while harnessing the attributes that have long ensured its transparency,” they wrote.
This latest proposal follows a suite of FDA draft guidances on gene therapies issued in July that proposed new guidance on manufacturing issues, long-term follow-up, and pathways for clinical development in certain areas.
The guidance is scheduled for publication in the Federal Register on Aug. 17.
SOURCE: Collins FS and Gottlieb S. N Engl J Med. doi: 10.1056/NEJMp1810628.
Key clinical point: NIH and FDA are looking to streamline gene therapy development oversight.
Major finding: The proposal would return the function of the Recombinant DNA Advisory Committee (RAC) to a function more in line with its original mission.
Study details: Full proposal details are to be published in the Federal Register on Aug. 17.
Disclosures: The agency leaders reported no relevant disclosures in the production of the proposal.
Source: Collins FS and Gottlieb S. N Engl J Med. doi: 10.1056/NEJMp1810628.
Health Canada approves emicizumab
Health Canada has approved emicizumab (Hemlibra®) for use as routine prophylaxis to prevent or reduce bleeding episodes in hemophilia A patients with factor VIII inhibitors.
Emicizumab is a bispecific factor IXa- and factor X-directed antibody. It bridges activated factor IX and factor X to restore the natural function of missing activated factor VIII that is needed for effective blood clotting.
Emicizumab is given as a once-weekly subcutaneous injection.
“Preventing bleeds in patients with hemophilia A can be extremely challenging, usually requiring patients to self-infuse medications multiple times a week, or even daily,” said Jayson Stoffman, MD, of the University of Manitoba in Winnipeg.
“The development of inhibitors adds a significant challenge, with more demanding treatments that are often less effective. Hemlibra offers these patients the chance to effectively reduce the frequency of their bleeds with a once-weekly injection at home. This could significantly improve the quality of life for inhibitor patients, and particularly children and their families.”
Trial results
The Health Canada approval of emicizumab is based on data from a pair of phase 3 trials—HAVEN 1 and HAVEN 2.
Results from HAVEN 1 were published in NEJM and presented at the 26th ISTH Congress in July 2017. Updated results from HAVEN 2 were presented at the 2017 ASH Annual Meeting in December.
HAVEN 1
This study enrolled 109 patients (age 12 and older) with hemophilia A and factor VIII inhibitors who were previously treated with bypassing agents (BPAs) on demand or as prophylaxis.
The patients were randomized to receive emicizumab prophylaxis or no prophylaxis. On-demand treatment of breakthrough bleeds with BPAs was allowed.
There was an 87% reduction in treated bleeds with emicizumab compared to no prophylaxis (P<0.0001). And there was an 80% reduction in all bleeds with emicizumab (P<0.0001).
The proportion of patients with 0 treated bleeds was 62.9% among emicizumab recipients and 5.6% among patients who did not receive prophylaxis.
Adverse events (AEs) occurring in at least 5% of patients treated with emicizumab were local injection site reactions, headache, fatigue, upper respiratory tract infection, and arthralgia.
Two patients experienced thromboembolic events (TEs), and 3 had thrombotic microangiopathy (TMA) while receiving emicizumab prophylaxis and more than 100 µ/kg/day of activated prothrombin complex concentrate, on average, for 24 hours or more before the event. Two of these patients had also received recombinant factor VIIa.
Neither TE required anticoagulation therapy, and 1 patient restarted emicizumab. The cases of TMA observed were transient, and 1 patient restarted emicizumab.
There was 1 death, but it was considered unrelated to emicizumab. The patient had developed TMA but died of rectal hemorrhage.
HAVEN 2
In this single-arm trial, researchers evaluated emicizumab prophylaxis in 60 patients, ages 1 to 17, who had hemophilia A with factor VIII inhibitors.
The efficacy analysis included 57 patients who were younger than 12. The 3 older patients were only included in the safety analysis.
Of the 57 patients, 64.9% had 0 bleeds, 94.7% had 0 treated bleeds, and 98.2% had 0 treated spontaneous bleeds/treated joint bleeds. None of the patients had treated target joint bleeds.
A subset of 23 patients received emicizumab for at least 12 weeks (median treatment duration of 38.1 weeks; range, 12.7 to 41.6 weeks).
Of these 23 patients, 34.8% had 0 bleeds, 87.0% had 0 treated bleeds, and 95.7% had 0 treated spontaneous bleeds/treated joint bleeds.
There were 40 patients who had a total of 201 AEs. The most common of these were viral upper respiratory tract infections (16.7%) and injection site reactions (16.7%).
There were no TEs or TMA events, and none of the patients tested positive for anti-drug antibodies. None of the 7 serious AEs in this trial were considered treatment-related.
Health Canada has approved emicizumab (Hemlibra®) for use as routine prophylaxis to prevent or reduce bleeding episodes in hemophilia A patients with factor VIII inhibitors.
Emicizumab is a bispecific factor IXa- and factor X-directed antibody. It bridges activated factor IX and factor X to restore the natural function of missing activated factor VIII that is needed for effective blood clotting.
Emicizumab is given as a once-weekly subcutaneous injection.
“Preventing bleeds in patients with hemophilia A can be extremely challenging, usually requiring patients to self-infuse medications multiple times a week, or even daily,” said Jayson Stoffman, MD, of the University of Manitoba in Winnipeg.
“The development of inhibitors adds a significant challenge, with more demanding treatments that are often less effective. Hemlibra offers these patients the chance to effectively reduce the frequency of their bleeds with a once-weekly injection at home. This could significantly improve the quality of life for inhibitor patients, and particularly children and their families.”
Trial results
The Health Canada approval of emicizumab is based on data from a pair of phase 3 trials—HAVEN 1 and HAVEN 2.
Results from HAVEN 1 were published in NEJM and presented at the 26th ISTH Congress in July 2017. Updated results from HAVEN 2 were presented at the 2017 ASH Annual Meeting in December.
HAVEN 1
This study enrolled 109 patients (age 12 and older) with hemophilia A and factor VIII inhibitors who were previously treated with bypassing agents (BPAs) on demand or as prophylaxis.
The patients were randomized to receive emicizumab prophylaxis or no prophylaxis. On-demand treatment of breakthrough bleeds with BPAs was allowed.
There was an 87% reduction in treated bleeds with emicizumab compared to no prophylaxis (P<0.0001). And there was an 80% reduction in all bleeds with emicizumab (P<0.0001).
The proportion of patients with 0 treated bleeds was 62.9% among emicizumab recipients and 5.6% among patients who did not receive prophylaxis.
Adverse events (AEs) occurring in at least 5% of patients treated with emicizumab were local injection site reactions, headache, fatigue, upper respiratory tract infection, and arthralgia.
Two patients experienced thromboembolic events (TEs), and 3 had thrombotic microangiopathy (TMA) while receiving emicizumab prophylaxis and more than 100 µ/kg/day of activated prothrombin complex concentrate, on average, for 24 hours or more before the event. Two of these patients had also received recombinant factor VIIa.
Neither TE required anticoagulation therapy, and 1 patient restarted emicizumab. The cases of TMA observed were transient, and 1 patient restarted emicizumab.
There was 1 death, but it was considered unrelated to emicizumab. The patient had developed TMA but died of rectal hemorrhage.
HAVEN 2
In this single-arm trial, researchers evaluated emicizumab prophylaxis in 60 patients, ages 1 to 17, who had hemophilia A with factor VIII inhibitors.
The efficacy analysis included 57 patients who were younger than 12. The 3 older patients were only included in the safety analysis.
Of the 57 patients, 64.9% had 0 bleeds, 94.7% had 0 treated bleeds, and 98.2% had 0 treated spontaneous bleeds/treated joint bleeds. None of the patients had treated target joint bleeds.
A subset of 23 patients received emicizumab for at least 12 weeks (median treatment duration of 38.1 weeks; range, 12.7 to 41.6 weeks).
Of these 23 patients, 34.8% had 0 bleeds, 87.0% had 0 treated bleeds, and 95.7% had 0 treated spontaneous bleeds/treated joint bleeds.
There were 40 patients who had a total of 201 AEs. The most common of these were viral upper respiratory tract infections (16.7%) and injection site reactions (16.7%).
There were no TEs or TMA events, and none of the patients tested positive for anti-drug antibodies. None of the 7 serious AEs in this trial were considered treatment-related.
Health Canada has approved emicizumab (Hemlibra®) for use as routine prophylaxis to prevent or reduce bleeding episodes in hemophilia A patients with factor VIII inhibitors.
Emicizumab is a bispecific factor IXa- and factor X-directed antibody. It bridges activated factor IX and factor X to restore the natural function of missing activated factor VIII that is needed for effective blood clotting.
Emicizumab is given as a once-weekly subcutaneous injection.
“Preventing bleeds in patients with hemophilia A can be extremely challenging, usually requiring patients to self-infuse medications multiple times a week, or even daily,” said Jayson Stoffman, MD, of the University of Manitoba in Winnipeg.
“The development of inhibitors adds a significant challenge, with more demanding treatments that are often less effective. Hemlibra offers these patients the chance to effectively reduce the frequency of their bleeds with a once-weekly injection at home. This could significantly improve the quality of life for inhibitor patients, and particularly children and their families.”
Trial results
The Health Canada approval of emicizumab is based on data from a pair of phase 3 trials—HAVEN 1 and HAVEN 2.
Results from HAVEN 1 were published in NEJM and presented at the 26th ISTH Congress in July 2017. Updated results from HAVEN 2 were presented at the 2017 ASH Annual Meeting in December.
HAVEN 1
This study enrolled 109 patients (age 12 and older) with hemophilia A and factor VIII inhibitors who were previously treated with bypassing agents (BPAs) on demand or as prophylaxis.
The patients were randomized to receive emicizumab prophylaxis or no prophylaxis. On-demand treatment of breakthrough bleeds with BPAs was allowed.
There was an 87% reduction in treated bleeds with emicizumab compared to no prophylaxis (P<0.0001). And there was an 80% reduction in all bleeds with emicizumab (P<0.0001).
The proportion of patients with 0 treated bleeds was 62.9% among emicizumab recipients and 5.6% among patients who did not receive prophylaxis.
Adverse events (AEs) occurring in at least 5% of patients treated with emicizumab were local injection site reactions, headache, fatigue, upper respiratory tract infection, and arthralgia.
Two patients experienced thromboembolic events (TEs), and 3 had thrombotic microangiopathy (TMA) while receiving emicizumab prophylaxis and more than 100 µ/kg/day of activated prothrombin complex concentrate, on average, for 24 hours or more before the event. Two of these patients had also received recombinant factor VIIa.
Neither TE required anticoagulation therapy, and 1 patient restarted emicizumab. The cases of TMA observed were transient, and 1 patient restarted emicizumab.
There was 1 death, but it was considered unrelated to emicizumab. The patient had developed TMA but died of rectal hemorrhage.
HAVEN 2
In this single-arm trial, researchers evaluated emicizumab prophylaxis in 60 patients, ages 1 to 17, who had hemophilia A with factor VIII inhibitors.
The efficacy analysis included 57 patients who were younger than 12. The 3 older patients were only included in the safety analysis.
Of the 57 patients, 64.9% had 0 bleeds, 94.7% had 0 treated bleeds, and 98.2% had 0 treated spontaneous bleeds/treated joint bleeds. None of the patients had treated target joint bleeds.
A subset of 23 patients received emicizumab for at least 12 weeks (median treatment duration of 38.1 weeks; range, 12.7 to 41.6 weeks).
Of these 23 patients, 34.8% had 0 bleeds, 87.0% had 0 treated bleeds, and 95.7% had 0 treated spontaneous bleeds/treated joint bleeds.
There were 40 patients who had a total of 201 AEs. The most common of these were viral upper respiratory tract infections (16.7%) and injection site reactions (16.7%).
There were no TEs or TMA events, and none of the patients tested positive for anti-drug antibodies. None of the 7 serious AEs in this trial were considered treatment-related.
FDA approves second drug for thrombocytopenia in liver disease
The Food and Drug Administration has approved lusutrombopag (Mulpleta) for the treatment of thrombocytopenia in patients with chronic liver disease who are scheduled to undergo medical or dental procedures. The drug is expected to be available in the United States in September 2018.
In May 2018, the FDA approved avatrombopag (Doptelet) as the first treatment for thrombocytopenia in this patient population.
The approval is based on a pair of phase 3, double-blind, placebo-controlled clinical trials – L-PLUS 1 and L-PLUS 2 (NCT02389621) – that included 312 patients who have chronic liver disease with severe thrombocytopenia. In L-PLUS 1, the primary endpoint was the percentage of patients who didn’t need platelet transfusions before the primary procedure was performed; this was met by 78% in the lusutrombopag group versus just 13% in the control group. In L-PLUS 2, the primary endpoint was the percentage of patients who didn’t need platelet transfusions before the procedure and didn’t require rescue therapy from randomization through 7 days post procedure; this was met by 65% of patients the lusutrombopag group versus 29% of patients the control group.
The most common adverse event was headache, and the recommended dosage for lusutrombopag is 3 mg orally once daily with or without food for 7 days. Lusutrombopag is not indicated for general platelet normalization in patients with chronic liver disease and thrombocytopenia. Full prescribing information and further information about the approval can be found on the FDA website.
Lusutrombopag was approved in Japan in 2015 for this indication and is slated to be evaluated by the European Medicines Agency in 2019. The drug is marketed by Shionogi.
The Food and Drug Administration has approved lusutrombopag (Mulpleta) for the treatment of thrombocytopenia in patients with chronic liver disease who are scheduled to undergo medical or dental procedures. The drug is expected to be available in the United States in September 2018.
In May 2018, the FDA approved avatrombopag (Doptelet) as the first treatment for thrombocytopenia in this patient population.
The approval is based on a pair of phase 3, double-blind, placebo-controlled clinical trials – L-PLUS 1 and L-PLUS 2 (NCT02389621) – that included 312 patients who have chronic liver disease with severe thrombocytopenia. In L-PLUS 1, the primary endpoint was the percentage of patients who didn’t need platelet transfusions before the primary procedure was performed; this was met by 78% in the lusutrombopag group versus just 13% in the control group. In L-PLUS 2, the primary endpoint was the percentage of patients who didn’t need platelet transfusions before the procedure and didn’t require rescue therapy from randomization through 7 days post procedure; this was met by 65% of patients the lusutrombopag group versus 29% of patients the control group.
The most common adverse event was headache, and the recommended dosage for lusutrombopag is 3 mg orally once daily with or without food for 7 days. Lusutrombopag is not indicated for general platelet normalization in patients with chronic liver disease and thrombocytopenia. Full prescribing information and further information about the approval can be found on the FDA website.
Lusutrombopag was approved in Japan in 2015 for this indication and is slated to be evaluated by the European Medicines Agency in 2019. The drug is marketed by Shionogi.
The Food and Drug Administration has approved lusutrombopag (Mulpleta) for the treatment of thrombocytopenia in patients with chronic liver disease who are scheduled to undergo medical or dental procedures. The drug is expected to be available in the United States in September 2018.
In May 2018, the FDA approved avatrombopag (Doptelet) as the first treatment for thrombocytopenia in this patient population.
The approval is based on a pair of phase 3, double-blind, placebo-controlled clinical trials – L-PLUS 1 and L-PLUS 2 (NCT02389621) – that included 312 patients who have chronic liver disease with severe thrombocytopenia. In L-PLUS 1, the primary endpoint was the percentage of patients who didn’t need platelet transfusions before the primary procedure was performed; this was met by 78% in the lusutrombopag group versus just 13% in the control group. In L-PLUS 2, the primary endpoint was the percentage of patients who didn’t need platelet transfusions before the procedure and didn’t require rescue therapy from randomization through 7 days post procedure; this was met by 65% of patients the lusutrombopag group versus 29% of patients the control group.
The most common adverse event was headache, and the recommended dosage for lusutrombopag is 3 mg orally once daily with or without food for 7 days. Lusutrombopag is not indicated for general platelet normalization in patients with chronic liver disease and thrombocytopenia. Full prescribing information and further information about the approval can be found on the FDA website.
Lusutrombopag was approved in Japan in 2015 for this indication and is slated to be evaluated by the European Medicines Agency in 2019. The drug is marketed by Shionogi.
FA pathway key to DNA repair after CRISPR cutting
New research suggests the Fanconi anemia (FA) pathway plays a key role in repairing double-strand breaks (DSBs) created by CRISPR-Cas9 genome editing.
Researchers said they found that Cas9-induced single-strand template repair requires the FA pathway, and the protein FANCD2 localizes to Cas9-induced DSBs.
The team said this research provides insight into why CRISPR-Cas9 does not produce equal success in all cells.
Furthermore, the work might help researchers boost the efficiency with which cells insert new DNA into the genome and generally tweak CRISPR-Cas9 editing to get the desired outcome.
“If you want to treat sickle cell anemia, your chances of success are inextricably tied to the efficiency with which you can replace the mutated sickle cell gene with the correct one,” said study author Chris Richardson, PhD, formerly of the University of California, Berkeley, but now at Spotlight Therapeutics in Hayward, California.
“If you harvest a million cells from a patient and you have a 10% insertion rate, that is not as good as if you have 30% to 40%. Being able to manipulate those cells to increase the frequency of this process, called homology-directed repair, is exciting.”
Dr Richardson and his colleagues described this work in Nature Genetics.
CRISPR relies on DNA repair
The researchers noted that CRISPR-Cas9 creates targeted DSBs, and it’s up to the cell to repair the DNA.
This can happen in 2 ways. Enzymes can stitch the dangling ends back together, which often results in one or more bases being added or deleted, disrupting the function of the gene.
Alternatively, other enzymes can patch the break with a single strand of DNA that matches the DNA sequence upstream and downstream of the cut. A complementary DNA strand is created to complete the double-strand repair.
The former method, called non-homologous end-joining, appears to be the most common outcome after CRISPR cutting.
The latter method, homology-directed repair, happens more frequently in some cells than others and requires the presence of DNA that can be used to patch the break. Researchers often supply a single-stranded piece of DNA and hope the cell uses it to replace the faulty sequence with the new one.
Both processes are a bit mysterious, however, and no one knows why some cells readily patch in DNA while others do so infrequently.
“The enthusiasm for using CRISPR-Cas9 for medical or synthetic biology applications is great, but no one really knows what happens after you put it into cells,” Dr Richardson said. “It goes and creates these breaks, and you count on the cells to fix them, but people don’t really understand how that process works.”
To find out which DNA repair enzymes are critical to homology-directed repair after CRISPR cutting, Dr Richardson and his colleagues used a technique called CRISPR interference. They knocked out, one at a time, more than 2000 genes known or suspected to be involved in DNA repair.
The researchers were surprised to find that many of the genes that proved important—homology-directed repair dropped dramatically when they were silenced—were involved in the FA pathway.
FA pathway
The FA pathway was largely understood to repair DNA interstrand crosslinks, where a nucleotide on one strand of DNA bonds tightly with a nucleotide on the adjacent strand, interfering with DNA replication and often killing the cell.
“Based on our work, we believe that the Fanconi anemia pathway plays a major role in fixing other types of lesions as well, but is best understood as the pathway that repairs double-strand breaks,” Dr Richardson said. “After Cas9 editing, the Fanconi anemia pathway is required if you want to insert new DNA.”
The importance of the FA pathway in repairing DSBs casts doubt on some planned CRISPR treatments for Fanconi anemia itself.
Without an active FA pathway, cells may not be able to replace their mutated genes with normal genes after Cas9 makes a cut. In fact, the activity level of the FA pathway may affect how efficiently CRISPR can insert DNA in a specific cell.
The researchers concluded that, while end-joining is the default repair mechanism after a DSB, the FA pathway competes with it, and that higher activity results in more homology-directed repair and less end-joining.
Dr Richardson and his colleagues also found that 1 of the 21 proteins in the FA pathway, FANCD2, always homes in on the site of the DSB created by CRISPR-Cas9. This suggests FANCD2 plays an important role in regulating the insertion of new DNA at the cut site.
The researchers therefore believe FANCD2 could be tweaked to boost the frequency with which a cell inserts DNA via homology-directed repair.
“Also, since FANCD2 localizes to the site of Cas9 breaks, you can use FANCD2 to map where Cas9 is cutting in any cell type,” Dr Richardson said. “If you edit a population of cells and you want to know where the on- and off-target cuts are, you can just map where FANCD2 was found in the genome and you can find the cuts.”
New research suggests the Fanconi anemia (FA) pathway plays a key role in repairing double-strand breaks (DSBs) created by CRISPR-Cas9 genome editing.
Researchers said they found that Cas9-induced single-strand template repair requires the FA pathway, and the protein FANCD2 localizes to Cas9-induced DSBs.
The team said this research provides insight into why CRISPR-Cas9 does not produce equal success in all cells.
Furthermore, the work might help researchers boost the efficiency with which cells insert new DNA into the genome and generally tweak CRISPR-Cas9 editing to get the desired outcome.
“If you want to treat sickle cell anemia, your chances of success are inextricably tied to the efficiency with which you can replace the mutated sickle cell gene with the correct one,” said study author Chris Richardson, PhD, formerly of the University of California, Berkeley, but now at Spotlight Therapeutics in Hayward, California.
“If you harvest a million cells from a patient and you have a 10% insertion rate, that is not as good as if you have 30% to 40%. Being able to manipulate those cells to increase the frequency of this process, called homology-directed repair, is exciting.”
Dr Richardson and his colleagues described this work in Nature Genetics.
CRISPR relies on DNA repair
The researchers noted that CRISPR-Cas9 creates targeted DSBs, and it’s up to the cell to repair the DNA.
This can happen in 2 ways. Enzymes can stitch the dangling ends back together, which often results in one or more bases being added or deleted, disrupting the function of the gene.
Alternatively, other enzymes can patch the break with a single strand of DNA that matches the DNA sequence upstream and downstream of the cut. A complementary DNA strand is created to complete the double-strand repair.
The former method, called non-homologous end-joining, appears to be the most common outcome after CRISPR cutting.
The latter method, homology-directed repair, happens more frequently in some cells than others and requires the presence of DNA that can be used to patch the break. Researchers often supply a single-stranded piece of DNA and hope the cell uses it to replace the faulty sequence with the new one.
Both processes are a bit mysterious, however, and no one knows why some cells readily patch in DNA while others do so infrequently.
“The enthusiasm for using CRISPR-Cas9 for medical or synthetic biology applications is great, but no one really knows what happens after you put it into cells,” Dr Richardson said. “It goes and creates these breaks, and you count on the cells to fix them, but people don’t really understand how that process works.”
To find out which DNA repair enzymes are critical to homology-directed repair after CRISPR cutting, Dr Richardson and his colleagues used a technique called CRISPR interference. They knocked out, one at a time, more than 2000 genes known or suspected to be involved in DNA repair.
The researchers were surprised to find that many of the genes that proved important—homology-directed repair dropped dramatically when they were silenced—were involved in the FA pathway.
FA pathway
The FA pathway was largely understood to repair DNA interstrand crosslinks, where a nucleotide on one strand of DNA bonds tightly with a nucleotide on the adjacent strand, interfering with DNA replication and often killing the cell.
“Based on our work, we believe that the Fanconi anemia pathway plays a major role in fixing other types of lesions as well, but is best understood as the pathway that repairs double-strand breaks,” Dr Richardson said. “After Cas9 editing, the Fanconi anemia pathway is required if you want to insert new DNA.”
The importance of the FA pathway in repairing DSBs casts doubt on some planned CRISPR treatments for Fanconi anemia itself.
Without an active FA pathway, cells may not be able to replace their mutated genes with normal genes after Cas9 makes a cut. In fact, the activity level of the FA pathway may affect how efficiently CRISPR can insert DNA in a specific cell.
The researchers concluded that, while end-joining is the default repair mechanism after a DSB, the FA pathway competes with it, and that higher activity results in more homology-directed repair and less end-joining.
Dr Richardson and his colleagues also found that 1 of the 21 proteins in the FA pathway, FANCD2, always homes in on the site of the DSB created by CRISPR-Cas9. This suggests FANCD2 plays an important role in regulating the insertion of new DNA at the cut site.
The researchers therefore believe FANCD2 could be tweaked to boost the frequency with which a cell inserts DNA via homology-directed repair.
“Also, since FANCD2 localizes to the site of Cas9 breaks, you can use FANCD2 to map where Cas9 is cutting in any cell type,” Dr Richardson said. “If you edit a population of cells and you want to know where the on- and off-target cuts are, you can just map where FANCD2 was found in the genome and you can find the cuts.”
New research suggests the Fanconi anemia (FA) pathway plays a key role in repairing double-strand breaks (DSBs) created by CRISPR-Cas9 genome editing.
Researchers said they found that Cas9-induced single-strand template repair requires the FA pathway, and the protein FANCD2 localizes to Cas9-induced DSBs.
The team said this research provides insight into why CRISPR-Cas9 does not produce equal success in all cells.
Furthermore, the work might help researchers boost the efficiency with which cells insert new DNA into the genome and generally tweak CRISPR-Cas9 editing to get the desired outcome.
“If you want to treat sickle cell anemia, your chances of success are inextricably tied to the efficiency with which you can replace the mutated sickle cell gene with the correct one,” said study author Chris Richardson, PhD, formerly of the University of California, Berkeley, but now at Spotlight Therapeutics in Hayward, California.
“If you harvest a million cells from a patient and you have a 10% insertion rate, that is not as good as if you have 30% to 40%. Being able to manipulate those cells to increase the frequency of this process, called homology-directed repair, is exciting.”
Dr Richardson and his colleagues described this work in Nature Genetics.
CRISPR relies on DNA repair
The researchers noted that CRISPR-Cas9 creates targeted DSBs, and it’s up to the cell to repair the DNA.
This can happen in 2 ways. Enzymes can stitch the dangling ends back together, which often results in one or more bases being added or deleted, disrupting the function of the gene.
Alternatively, other enzymes can patch the break with a single strand of DNA that matches the DNA sequence upstream and downstream of the cut. A complementary DNA strand is created to complete the double-strand repair.
The former method, called non-homologous end-joining, appears to be the most common outcome after CRISPR cutting.
The latter method, homology-directed repair, happens more frequently in some cells than others and requires the presence of DNA that can be used to patch the break. Researchers often supply a single-stranded piece of DNA and hope the cell uses it to replace the faulty sequence with the new one.
Both processes are a bit mysterious, however, and no one knows why some cells readily patch in DNA while others do so infrequently.
“The enthusiasm for using CRISPR-Cas9 for medical or synthetic biology applications is great, but no one really knows what happens after you put it into cells,” Dr Richardson said. “It goes and creates these breaks, and you count on the cells to fix them, but people don’t really understand how that process works.”
To find out which DNA repair enzymes are critical to homology-directed repair after CRISPR cutting, Dr Richardson and his colleagues used a technique called CRISPR interference. They knocked out, one at a time, more than 2000 genes known or suspected to be involved in DNA repair.
The researchers were surprised to find that many of the genes that proved important—homology-directed repair dropped dramatically when they were silenced—were involved in the FA pathway.
FA pathway
The FA pathway was largely understood to repair DNA interstrand crosslinks, where a nucleotide on one strand of DNA bonds tightly with a nucleotide on the adjacent strand, interfering with DNA replication and often killing the cell.
“Based on our work, we believe that the Fanconi anemia pathway plays a major role in fixing other types of lesions as well, but is best understood as the pathway that repairs double-strand breaks,” Dr Richardson said. “After Cas9 editing, the Fanconi anemia pathway is required if you want to insert new DNA.”
The importance of the FA pathway in repairing DSBs casts doubt on some planned CRISPR treatments for Fanconi anemia itself.
Without an active FA pathway, cells may not be able to replace their mutated genes with normal genes after Cas9 makes a cut. In fact, the activity level of the FA pathway may affect how efficiently CRISPR can insert DNA in a specific cell.
The researchers concluded that, while end-joining is the default repair mechanism after a DSB, the FA pathway competes with it, and that higher activity results in more homology-directed repair and less end-joining.
Dr Richardson and his colleagues also found that 1 of the 21 proteins in the FA pathway, FANCD2, always homes in on the site of the DSB created by CRISPR-Cas9. This suggests FANCD2 plays an important role in regulating the insertion of new DNA at the cut site.
The researchers therefore believe FANCD2 could be tweaked to boost the frequency with which a cell inserts DNA via homology-directed repair.
“Also, since FANCD2 localizes to the site of Cas9 breaks, you can use FANCD2 to map where Cas9 is cutting in any cell type,” Dr Richardson said. “If you edit a population of cells and you want to know where the on- and off-target cuts are, you can just map where FANCD2 was found in the genome and you can find the cuts.”
Researchers identify potential sickle cell disease target
Researchers have identified a repressor of fetal hemoglobin (HbF), which they assert could offer a therapeutic target for patients with sickle cell disease (SCD) and some forms of beta thalassemia, according to a new paper published in Science.
Strategies to boost HbF have so far been limited to gene therapy and hydroxyurea, which has been shown to have limited efficacy.
The finding was made using a tailored CRISPR screen of adult human erythroid cells; the CRISPR screen targeted protein kinases, which are controllable by small molecules, which makes them more feasible for treatment therapeutically, researchers wrote.
“Using an improved CRISPR-Cas9 domain-focused screening approach, we identified the erythroid-specific kinase HRI [heme-regulated inhibitor] as a potentially druggable target that is involved in HbF silencing,” wrote Gerd A. Blobel, MD, PhD, of Children’s Hospital of Philadelphia and his colleagues.
HRI is an erythroid-specific kinase that controls protein translation. Once identified, depleting HRI resulted in an increase of HbF production and reduced sickling in cultured erythroid cells. Researchers said that diminished expression of the transcription factor BCL11A, an HbF repressor, accounted for the effects of HRI depletion.
The results with erythroid cell cultures suggest that HRI loss is well tolerated, but the mechanism of inducing fetal hemoglobin is still not fully understood, the researchers noted. Also, while HRI may be targetable, the extent of the benefits of this targeting are not yet known.
“It remains to be seen whether HRI inhibition in SCD patients would elevate HbF levels sufficiently to improve outcomes,” the researchers wrote. “HRI inhibition elevated HbF levels to a point at which it reduced cell sickling in culture, suggesting that pharmacologic HRI inhibitors may provide clinical benefit in SCD patients. Moreover, in light of our results, combining HRI inhibition with an additional pharmacologic HbF inducer may improve the therapeutic index. "Funding was provided by NIH training grants and Cold Spring Harbor Laboratory. Three of the authors reported that they are inventors on a patent submitted by the Children’s Hospital of Philadelphia that covers the therapeutic targeting of HRI for hemoglobinopathies.
SOURCE: Grevet J et al. Science. 2018 Jul 20. doi: 10.1126/science.aao0932.
Researchers have identified a repressor of fetal hemoglobin (HbF), which they assert could offer a therapeutic target for patients with sickle cell disease (SCD) and some forms of beta thalassemia, according to a new paper published in Science.
Strategies to boost HbF have so far been limited to gene therapy and hydroxyurea, which has been shown to have limited efficacy.
The finding was made using a tailored CRISPR screen of adult human erythroid cells; the CRISPR screen targeted protein kinases, which are controllable by small molecules, which makes them more feasible for treatment therapeutically, researchers wrote.
“Using an improved CRISPR-Cas9 domain-focused screening approach, we identified the erythroid-specific kinase HRI [heme-regulated inhibitor] as a potentially druggable target that is involved in HbF silencing,” wrote Gerd A. Blobel, MD, PhD, of Children’s Hospital of Philadelphia and his colleagues.
HRI is an erythroid-specific kinase that controls protein translation. Once identified, depleting HRI resulted in an increase of HbF production and reduced sickling in cultured erythroid cells. Researchers said that diminished expression of the transcription factor BCL11A, an HbF repressor, accounted for the effects of HRI depletion.
The results with erythroid cell cultures suggest that HRI loss is well tolerated, but the mechanism of inducing fetal hemoglobin is still not fully understood, the researchers noted. Also, while HRI may be targetable, the extent of the benefits of this targeting are not yet known.
“It remains to be seen whether HRI inhibition in SCD patients would elevate HbF levels sufficiently to improve outcomes,” the researchers wrote. “HRI inhibition elevated HbF levels to a point at which it reduced cell sickling in culture, suggesting that pharmacologic HRI inhibitors may provide clinical benefit in SCD patients. Moreover, in light of our results, combining HRI inhibition with an additional pharmacologic HbF inducer may improve the therapeutic index. "Funding was provided by NIH training grants and Cold Spring Harbor Laboratory. Three of the authors reported that they are inventors on a patent submitted by the Children’s Hospital of Philadelphia that covers the therapeutic targeting of HRI for hemoglobinopathies.
SOURCE: Grevet J et al. Science. 2018 Jul 20. doi: 10.1126/science.aao0932.
Researchers have identified a repressor of fetal hemoglobin (HbF), which they assert could offer a therapeutic target for patients with sickle cell disease (SCD) and some forms of beta thalassemia, according to a new paper published in Science.
Strategies to boost HbF have so far been limited to gene therapy and hydroxyurea, which has been shown to have limited efficacy.
The finding was made using a tailored CRISPR screen of adult human erythroid cells; the CRISPR screen targeted protein kinases, which are controllable by small molecules, which makes them more feasible for treatment therapeutically, researchers wrote.
“Using an improved CRISPR-Cas9 domain-focused screening approach, we identified the erythroid-specific kinase HRI [heme-regulated inhibitor] as a potentially druggable target that is involved in HbF silencing,” wrote Gerd A. Blobel, MD, PhD, of Children’s Hospital of Philadelphia and his colleagues.
HRI is an erythroid-specific kinase that controls protein translation. Once identified, depleting HRI resulted in an increase of HbF production and reduced sickling in cultured erythroid cells. Researchers said that diminished expression of the transcription factor BCL11A, an HbF repressor, accounted for the effects of HRI depletion.
The results with erythroid cell cultures suggest that HRI loss is well tolerated, but the mechanism of inducing fetal hemoglobin is still not fully understood, the researchers noted. Also, while HRI may be targetable, the extent of the benefits of this targeting are not yet known.
“It remains to be seen whether HRI inhibition in SCD patients would elevate HbF levels sufficiently to improve outcomes,” the researchers wrote. “HRI inhibition elevated HbF levels to a point at which it reduced cell sickling in culture, suggesting that pharmacologic HRI inhibitors may provide clinical benefit in SCD patients. Moreover, in light of our results, combining HRI inhibition with an additional pharmacologic HbF inducer may improve the therapeutic index. "Funding was provided by NIH training grants and Cold Spring Harbor Laboratory. Three of the authors reported that they are inventors on a patent submitted by the Children’s Hospital of Philadelphia that covers the therapeutic targeting of HRI for hemoglobinopathies.
SOURCE: Grevet J et al. Science. 2018 Jul 20. doi: 10.1126/science.aao0932.
REPORTING FROM SCIENCE
For men with SCD and priapism, hypoxia may prompt RBC adhesion
WASHINGTON – For male patients with sickle cell disease, priapism can be more than just painful and embarrassing. The prolonged erections prompted by vasoocclusive events in the penis may lead to irreversible impotence, but little is known about risk factors for priapism, which remains a difficult-to-treat complication of the disease.
In males with HbSS sickle cell disease (SCD) and priapism, RBC adhesion is increased in hypoxic conditions, according to preliminary findings from work using a newly developed biochip that mimics microvascular conditions in SCD. This significant level of adhesion prompted by hypoxia was not seen in men who did not have priapism, according to study coauthor Erina Quinn, a research assistant in hematology and oncology at Case Western Reserve University, Cleveland, who presented the results at the annual meeting of the Foundation for Sickle Cell Disease Research.
When hemoglobin desaturation occurs, polymerization can be increased, leading to increased end-organ damage, Ms. Quinn said. The biochip is “an effort to measure cellular adhesion in a clinically meaningful way.” The tool can detect hemoglobin phenotype, differentiating among HbSS, HbSbeta+, and HbSC. It can also measure the degree of hemolysis and RBC deformability.
The biochip “mimics postcapillary flow conditions in microchannels,” Ms. Quinn said. The device forces blood samples through microchannels that are at the diameter of smaller venules, approximately 50 mcm, and at a physiological flow rate ranging from 1-13 mm/sec. The microfluidic channels are coated with laminin, a subendothelial matrix protein implicated in RBC adhesion. A second microfluidic biochip mimics hypoxic conditions.
The study enrolled 26 men with the HbSS genotype, 14 of whom reported priapism, and assessed RBC adhesion in blood samples run though both the SCD-modeled biochip and the hypoxia biochip. Investigators also assessed contemporaneous in vivo hemoglobin desaturation, and looked for associations with the in vitro biochip findings.
Of the 26 participants, 16 also had either nocturnal or exertional hemoglobin desaturation. In addition, 10 participants had both priapism and desaturations. These data were collected by retrospective chart review and patient survey.
Patients with priapism were a mean age of 34 years, compared with a mean age of 29 years for the other participants, a nonsignificant difference. There were no significant differences in mean hemoglobin or bilirubin levels, or in reticulocyte counts, between the two groups.
However, white blood count, absolute neutrophil count, and lactate dehydrogenase levels were significantly higher for men with priapism (P = .022, .037, and .008, respectively). Ferritin levels were higher as well, at a mean 2,433 (plus or minus 2,234) mcg/L for those with priapism, compared with a mean 269 (plus or minus 3,015) mcg/L for those without priapism (P = .031).
When absolute reticulocyte count was mapped against lactate dehydrogenase levels to create a measure of degree of hemolysis, “individuals with priapism had a more hemolytic lab profile,” said Ms. Quinn (P = .0186).
Though 10 of 14 men with priapism had hemoglobin desaturation, compared with 5 of 12 who did not have priapism, the difference was not statistically significant.
When the researchers compared microchip analysis of RBC adhesion, though, they found marked differences in RBC adhesion in hypoxic versus nonhypoxic conditions. Significantly more RBCs were adherent under hypoxic conditions – in the hypoxic biochip – for the patients with priapism than for patients without priapism (mean, 529 vs. 3,268 adherent cells; P = .016).
Though numbers were small, RBCs from patients with reported priapism and hemoglobin desaturation in vivo showed increased hypoxia enhanced adhesion in vitro (P = .013), Ms. Quinn said. These was no significant difference between adhesion in normoxic and hypoxic conditions for the patients without priapism.
Future directions of work with the biochip include prospective identification of desaturation events and better characterization of nocturnal symptoms, Ms. Quinn said. The investigators also plan to see whether treatment with supplemental oxygen affects RBC adhesion.
The research was supported by the National Institutes of Health, the Doris Duke Charitable Foundation, and the National Science Foundation. Two coauthors have filed an international patent for the biochip technology.
WASHINGTON – For male patients with sickle cell disease, priapism can be more than just painful and embarrassing. The prolonged erections prompted by vasoocclusive events in the penis may lead to irreversible impotence, but little is known about risk factors for priapism, which remains a difficult-to-treat complication of the disease.
In males with HbSS sickle cell disease (SCD) and priapism, RBC adhesion is increased in hypoxic conditions, according to preliminary findings from work using a newly developed biochip that mimics microvascular conditions in SCD. This significant level of adhesion prompted by hypoxia was not seen in men who did not have priapism, according to study coauthor Erina Quinn, a research assistant in hematology and oncology at Case Western Reserve University, Cleveland, who presented the results at the annual meeting of the Foundation for Sickle Cell Disease Research.
When hemoglobin desaturation occurs, polymerization can be increased, leading to increased end-organ damage, Ms. Quinn said. The biochip is “an effort to measure cellular adhesion in a clinically meaningful way.” The tool can detect hemoglobin phenotype, differentiating among HbSS, HbSbeta+, and HbSC. It can also measure the degree of hemolysis and RBC deformability.
The biochip “mimics postcapillary flow conditions in microchannels,” Ms. Quinn said. The device forces blood samples through microchannels that are at the diameter of smaller venules, approximately 50 mcm, and at a physiological flow rate ranging from 1-13 mm/sec. The microfluidic channels are coated with laminin, a subendothelial matrix protein implicated in RBC adhesion. A second microfluidic biochip mimics hypoxic conditions.
The study enrolled 26 men with the HbSS genotype, 14 of whom reported priapism, and assessed RBC adhesion in blood samples run though both the SCD-modeled biochip and the hypoxia biochip. Investigators also assessed contemporaneous in vivo hemoglobin desaturation, and looked for associations with the in vitro biochip findings.
Of the 26 participants, 16 also had either nocturnal or exertional hemoglobin desaturation. In addition, 10 participants had both priapism and desaturations. These data were collected by retrospective chart review and patient survey.
Patients with priapism were a mean age of 34 years, compared with a mean age of 29 years for the other participants, a nonsignificant difference. There were no significant differences in mean hemoglobin or bilirubin levels, or in reticulocyte counts, between the two groups.
However, white blood count, absolute neutrophil count, and lactate dehydrogenase levels were significantly higher for men with priapism (P = .022, .037, and .008, respectively). Ferritin levels were higher as well, at a mean 2,433 (plus or minus 2,234) mcg/L for those with priapism, compared with a mean 269 (plus or minus 3,015) mcg/L for those without priapism (P = .031).
When absolute reticulocyte count was mapped against lactate dehydrogenase levels to create a measure of degree of hemolysis, “individuals with priapism had a more hemolytic lab profile,” said Ms. Quinn (P = .0186).
Though 10 of 14 men with priapism had hemoglobin desaturation, compared with 5 of 12 who did not have priapism, the difference was not statistically significant.
When the researchers compared microchip analysis of RBC adhesion, though, they found marked differences in RBC adhesion in hypoxic versus nonhypoxic conditions. Significantly more RBCs were adherent under hypoxic conditions – in the hypoxic biochip – for the patients with priapism than for patients without priapism (mean, 529 vs. 3,268 adherent cells; P = .016).
Though numbers were small, RBCs from patients with reported priapism and hemoglobin desaturation in vivo showed increased hypoxia enhanced adhesion in vitro (P = .013), Ms. Quinn said. These was no significant difference between adhesion in normoxic and hypoxic conditions for the patients without priapism.
Future directions of work with the biochip include prospective identification of desaturation events and better characterization of nocturnal symptoms, Ms. Quinn said. The investigators also plan to see whether treatment with supplemental oxygen affects RBC adhesion.
The research was supported by the National Institutes of Health, the Doris Duke Charitable Foundation, and the National Science Foundation. Two coauthors have filed an international patent for the biochip technology.
WASHINGTON – For male patients with sickle cell disease, priapism can be more than just painful and embarrassing. The prolonged erections prompted by vasoocclusive events in the penis may lead to irreversible impotence, but little is known about risk factors for priapism, which remains a difficult-to-treat complication of the disease.
In males with HbSS sickle cell disease (SCD) and priapism, RBC adhesion is increased in hypoxic conditions, according to preliminary findings from work using a newly developed biochip that mimics microvascular conditions in SCD. This significant level of adhesion prompted by hypoxia was not seen in men who did not have priapism, according to study coauthor Erina Quinn, a research assistant in hematology and oncology at Case Western Reserve University, Cleveland, who presented the results at the annual meeting of the Foundation for Sickle Cell Disease Research.
When hemoglobin desaturation occurs, polymerization can be increased, leading to increased end-organ damage, Ms. Quinn said. The biochip is “an effort to measure cellular adhesion in a clinically meaningful way.” The tool can detect hemoglobin phenotype, differentiating among HbSS, HbSbeta+, and HbSC. It can also measure the degree of hemolysis and RBC deformability.
The biochip “mimics postcapillary flow conditions in microchannels,” Ms. Quinn said. The device forces blood samples through microchannels that are at the diameter of smaller venules, approximately 50 mcm, and at a physiological flow rate ranging from 1-13 mm/sec. The microfluidic channels are coated with laminin, a subendothelial matrix protein implicated in RBC adhesion. A second microfluidic biochip mimics hypoxic conditions.
The study enrolled 26 men with the HbSS genotype, 14 of whom reported priapism, and assessed RBC adhesion in blood samples run though both the SCD-modeled biochip and the hypoxia biochip. Investigators also assessed contemporaneous in vivo hemoglobin desaturation, and looked for associations with the in vitro biochip findings.
Of the 26 participants, 16 also had either nocturnal or exertional hemoglobin desaturation. In addition, 10 participants had both priapism and desaturations. These data were collected by retrospective chart review and patient survey.
Patients with priapism were a mean age of 34 years, compared with a mean age of 29 years for the other participants, a nonsignificant difference. There were no significant differences in mean hemoglobin or bilirubin levels, or in reticulocyte counts, between the two groups.
However, white blood count, absolute neutrophil count, and lactate dehydrogenase levels were significantly higher for men with priapism (P = .022, .037, and .008, respectively). Ferritin levels were higher as well, at a mean 2,433 (plus or minus 2,234) mcg/L for those with priapism, compared with a mean 269 (plus or minus 3,015) mcg/L for those without priapism (P = .031).
When absolute reticulocyte count was mapped against lactate dehydrogenase levels to create a measure of degree of hemolysis, “individuals with priapism had a more hemolytic lab profile,” said Ms. Quinn (P = .0186).
Though 10 of 14 men with priapism had hemoglobin desaturation, compared with 5 of 12 who did not have priapism, the difference was not statistically significant.
When the researchers compared microchip analysis of RBC adhesion, though, they found marked differences in RBC adhesion in hypoxic versus nonhypoxic conditions. Significantly more RBCs were adherent under hypoxic conditions – in the hypoxic biochip – for the patients with priapism than for patients without priapism (mean, 529 vs. 3,268 adherent cells; P = .016).
Though numbers were small, RBCs from patients with reported priapism and hemoglobin desaturation in vivo showed increased hypoxia enhanced adhesion in vitro (P = .013), Ms. Quinn said. These was no significant difference between adhesion in normoxic and hypoxic conditions for the patients without priapism.
Future directions of work with the biochip include prospective identification of desaturation events and better characterization of nocturnal symptoms, Ms. Quinn said. The investigators also plan to see whether treatment with supplemental oxygen affects RBC adhesion.
The research was supported by the National Institutes of Health, the Doris Duke Charitable Foundation, and the National Science Foundation. Two coauthors have filed an international patent for the biochip technology.
REPORTING FROM FSCDR 2018
Key clinical point: RBC adhesion was increased, but only in hypoxia, for men with sickle cell disease and priapism.
Major finding: Men who had desaturations and priapism had significantly higher RBC adhesion than those without priapism (P = .013).
Study details: An in vitro and in vivo study of 26 men with HbSS sickle cell disease, with and without priapism.
Disclosures: The study was funded by the National Institutes of Health, the Doris Duke Charitable Foundation, and the National Science Foundation. Two coauthors have filed an international patent for the biochip technology.
FDA issues guidance for hemophilia gene therapy development
The Food and Drug Administration issued its first-ever guidance on the development of human gene therapy for hemophilia.
The draft document provides a glimpse at the agency’s current thinking on clinical trial design and preclinical considerations in developing gene therapies. Specifically, it provides information to help companies set surrogate endpoints when seeking accelerated approval for hemophilia gene therapy products.
The hemophilia guidance is one of three disease-specific guidance documents. The other two clinical areas are retinal disorders and rare diseases.
FDA officials also issued updates to three more technical documents that deal with manufacturing of gene therapies.
The overall framework is similar to what FDA officials issued in 2017 for regenerative medicine. In a statement, FDA Commissioner Scott Gottlieb, MD, said the agency was responding to an increasing number of gene therapy approvals and anticipated development.
“Once just a theory, gene therapies are now a therapeutic reality for some patients,” Dr. Gottlieb said. “These platforms may have the potential to treat and cure some of our most intractable and vexing diseases. The policy framework we construct for how these products should be developed, reviewed by regulators, and reimbursed, will help set the stage for the continued advancement of this new market.”
The Food and Drug Administration issued its first-ever guidance on the development of human gene therapy for hemophilia.
The draft document provides a glimpse at the agency’s current thinking on clinical trial design and preclinical considerations in developing gene therapies. Specifically, it provides information to help companies set surrogate endpoints when seeking accelerated approval for hemophilia gene therapy products.
The hemophilia guidance is one of three disease-specific guidance documents. The other two clinical areas are retinal disorders and rare diseases.
FDA officials also issued updates to three more technical documents that deal with manufacturing of gene therapies.
The overall framework is similar to what FDA officials issued in 2017 for regenerative medicine. In a statement, FDA Commissioner Scott Gottlieb, MD, said the agency was responding to an increasing number of gene therapy approvals and anticipated development.
“Once just a theory, gene therapies are now a therapeutic reality for some patients,” Dr. Gottlieb said. “These platforms may have the potential to treat and cure some of our most intractable and vexing diseases. The policy framework we construct for how these products should be developed, reviewed by regulators, and reimbursed, will help set the stage for the continued advancement of this new market.”
The Food and Drug Administration issued its first-ever guidance on the development of human gene therapy for hemophilia.
The draft document provides a glimpse at the agency’s current thinking on clinical trial design and preclinical considerations in developing gene therapies. Specifically, it provides information to help companies set surrogate endpoints when seeking accelerated approval for hemophilia gene therapy products.
The hemophilia guidance is one of three disease-specific guidance documents. The other two clinical areas are retinal disorders and rare diseases.
FDA officials also issued updates to three more technical documents that deal with manufacturing of gene therapies.
The overall framework is similar to what FDA officials issued in 2017 for regenerative medicine. In a statement, FDA Commissioner Scott Gottlieb, MD, said the agency was responding to an increasing number of gene therapy approvals and anticipated development.
“Once just a theory, gene therapies are now a therapeutic reality for some patients,” Dr. Gottlieb said. “These platforms may have the potential to treat and cure some of our most intractable and vexing diseases. The policy framework we construct for how these products should be developed, reviewed by regulators, and reimbursed, will help set the stage for the continued advancement of this new market.”
Pfizer launches phase 3 gene therapy study in hemophilia B
Pfizer has begun early work on a phase 3 study of an investigational gene therapy to treat hemophilia B.
Pfizer, along with Spark Therapeutics, launched a phase 3 lead-in study at multiple centers to evaluate the efficacy and safety of current factor IX prophylaxis replacement therapy in the usual care setting. The efficacy data from the lead-in study will become the within-subject control group for patients who enroll in the next part of the
Fidanacogene elaparvovec is a vector containing a bioengineered adeno-associated virus capsid and a high-activity human coagulation factor IX gene. In an ongoing phase 1/2 trial of fidanacogene elaparvovec, all 15 participants with hemophilia B were able to discontinue routine infusion of factor IX concentrates without serious adverse events, according to data released by Pfizer and Spark Therapeutics in May 2018.
Pfizer has begun early work on a phase 3 study of an investigational gene therapy to treat hemophilia B.
Pfizer, along with Spark Therapeutics, launched a phase 3 lead-in study at multiple centers to evaluate the efficacy and safety of current factor IX prophylaxis replacement therapy in the usual care setting. The efficacy data from the lead-in study will become the within-subject control group for patients who enroll in the next part of the
Fidanacogene elaparvovec is a vector containing a bioengineered adeno-associated virus capsid and a high-activity human coagulation factor IX gene. In an ongoing phase 1/2 trial of fidanacogene elaparvovec, all 15 participants with hemophilia B were able to discontinue routine infusion of factor IX concentrates without serious adverse events, according to data released by Pfizer and Spark Therapeutics in May 2018.
Pfizer has begun early work on a phase 3 study of an investigational gene therapy to treat hemophilia B.
Pfizer, along with Spark Therapeutics, launched a phase 3 lead-in study at multiple centers to evaluate the efficacy and safety of current factor IX prophylaxis replacement therapy in the usual care setting. The efficacy data from the lead-in study will become the within-subject control group for patients who enroll in the next part of the
Fidanacogene elaparvovec is a vector containing a bioengineered adeno-associated virus capsid and a high-activity human coagulation factor IX gene. In an ongoing phase 1/2 trial of fidanacogene elaparvovec, all 15 participants with hemophilia B were able to discontinue routine infusion of factor IX concentrates without serious adverse events, according to data released by Pfizer and Spark Therapeutics in May 2018.
FDA releases guidance docs on gene therapy
The US Food and Drug Administration (FDA) has released several draft guidance documents on gene therapy.
Three are disease-specific guidances—for hemophilia, rare diseases, and retinal disorders—and 3 are guidances on manufacturing gene therapies.
These 6 documents are intended to serve as the building blocks of a modern, comprehensive framework for advancing the field of gene therapy, according to FDA Commissioner Scott Gottlieb, MD.
He said the documents are being issued in draft form so the FDA can solicit public input on these new policies. As with all draft guidances, all comments received will be considered before the FDA finalizes the documents.
Disease-specific guidances
The Human Gene Therapy for Rare Diseases Draft Guidance and the Human Gene Therapy for Retinal Disorders Draft Guidance include considerations for product development, preclinical research, clinical trials, expedited programs, and communication with the FDA.
The Human Gene Therapy for Hemophilia Draft Guidance covers the same topics but also includes considerations for measuring factor VIII and factor IX activity.
The draft guidance for rare diseases encompasses diseases affecting fewer than 200,000 people in the US.
Manufacturing guidances
The 3 remaining draft guidances are actually updates to existing guidances that address manufacturing issues related to gene therapy.
The first updated draft guidance, Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs), provides sponsors with recommendations on how to provide sufficient CMC information to ensure safety, identity, quality, purity, and strength/potency of investigational gene therapy products. This guidance applies to gene therapies alone and products that contain a gene therapy in combination with a drug or device.
The second draft guidance, Testing of Retroviral Vector-Based Gene Therapy Products for Replication Competent Retrovirus (RCR) during Product Manufacture and Patient Follow-up, provides recommendations regarding the proper testing for RCR during the manufacture of retroviral vector-based gene therapy products, as well as during the follow-up monitoring of patients who have received retroviral vector-based gene therapy products.
The third draft guidance, Long Term Follow-Up (LTFU) After Administration of Human Gene Therapy Products, provides recommendations regarding the design of LTFU observational studies for the collection of data on delayed adverse events after gene therapy.
Once finalized, these draft guidances will replace previous guidances issued by the FDA in April 2008 (CMC) and November 2006 (RCR and LTFU).
The US Food and Drug Administration (FDA) has released several draft guidance documents on gene therapy.
Three are disease-specific guidances—for hemophilia, rare diseases, and retinal disorders—and 3 are guidances on manufacturing gene therapies.
These 6 documents are intended to serve as the building blocks of a modern, comprehensive framework for advancing the field of gene therapy, according to FDA Commissioner Scott Gottlieb, MD.
He said the documents are being issued in draft form so the FDA can solicit public input on these new policies. As with all draft guidances, all comments received will be considered before the FDA finalizes the documents.
Disease-specific guidances
The Human Gene Therapy for Rare Diseases Draft Guidance and the Human Gene Therapy for Retinal Disorders Draft Guidance include considerations for product development, preclinical research, clinical trials, expedited programs, and communication with the FDA.
The Human Gene Therapy for Hemophilia Draft Guidance covers the same topics but also includes considerations for measuring factor VIII and factor IX activity.
The draft guidance for rare diseases encompasses diseases affecting fewer than 200,000 people in the US.
Manufacturing guidances
The 3 remaining draft guidances are actually updates to existing guidances that address manufacturing issues related to gene therapy.
The first updated draft guidance, Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs), provides sponsors with recommendations on how to provide sufficient CMC information to ensure safety, identity, quality, purity, and strength/potency of investigational gene therapy products. This guidance applies to gene therapies alone and products that contain a gene therapy in combination with a drug or device.
The second draft guidance, Testing of Retroviral Vector-Based Gene Therapy Products for Replication Competent Retrovirus (RCR) during Product Manufacture and Patient Follow-up, provides recommendations regarding the proper testing for RCR during the manufacture of retroviral vector-based gene therapy products, as well as during the follow-up monitoring of patients who have received retroviral vector-based gene therapy products.
The third draft guidance, Long Term Follow-Up (LTFU) After Administration of Human Gene Therapy Products, provides recommendations regarding the design of LTFU observational studies for the collection of data on delayed adverse events after gene therapy.
Once finalized, these draft guidances will replace previous guidances issued by the FDA in April 2008 (CMC) and November 2006 (RCR and LTFU).
The US Food and Drug Administration (FDA) has released several draft guidance documents on gene therapy.
Three are disease-specific guidances—for hemophilia, rare diseases, and retinal disorders—and 3 are guidances on manufacturing gene therapies.
These 6 documents are intended to serve as the building blocks of a modern, comprehensive framework for advancing the field of gene therapy, according to FDA Commissioner Scott Gottlieb, MD.
He said the documents are being issued in draft form so the FDA can solicit public input on these new policies. As with all draft guidances, all comments received will be considered before the FDA finalizes the documents.
Disease-specific guidances
The Human Gene Therapy for Rare Diseases Draft Guidance and the Human Gene Therapy for Retinal Disorders Draft Guidance include considerations for product development, preclinical research, clinical trials, expedited programs, and communication with the FDA.
The Human Gene Therapy for Hemophilia Draft Guidance covers the same topics but also includes considerations for measuring factor VIII and factor IX activity.
The draft guidance for rare diseases encompasses diseases affecting fewer than 200,000 people in the US.
Manufacturing guidances
The 3 remaining draft guidances are actually updates to existing guidances that address manufacturing issues related to gene therapy.
The first updated draft guidance, Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs), provides sponsors with recommendations on how to provide sufficient CMC information to ensure safety, identity, quality, purity, and strength/potency of investigational gene therapy products. This guidance applies to gene therapies alone and products that contain a gene therapy in combination with a drug or device.
The second draft guidance, Testing of Retroviral Vector-Based Gene Therapy Products for Replication Competent Retrovirus (RCR) during Product Manufacture and Patient Follow-up, provides recommendations regarding the proper testing for RCR during the manufacture of retroviral vector-based gene therapy products, as well as during the follow-up monitoring of patients who have received retroviral vector-based gene therapy products.
The third draft guidance, Long Term Follow-Up (LTFU) After Administration of Human Gene Therapy Products, provides recommendations regarding the design of LTFU observational studies for the collection of data on delayed adverse events after gene therapy.
Once finalized, these draft guidances will replace previous guidances issued by the FDA in April 2008 (CMC) and November 2006 (RCR and LTFU).
CHMP recommends rVWF for VWD
The European Medicines Agency’s Committee for Medicinal Products for Human Use (CHMP) has recommended marketing authorization for vonicog alfa (Veyvondi), a recombinant von Willebrand factor (rVWF) product.
The CHMP is recommending vonicog alfa for the treatment of bleeding events and treatment/prevention of surgical bleeding in adults (age 18 and older) with von Willebrand disease (VWD) when desmopressin treatment alone is ineffective or not indicated.
The CHMP’s recommendation will be reviewed by the European Commission, which has the authority to approve medicines for use in the European Union, Norway, Iceland, and Liechtenstein.
The European Commission usually makes a decision within 67 days of the CHMP’s recommendation.
The CHMP’s opinion of vonicog alfa is supported by a pair of phase 3 trials.
Non-surgical setting
Results from a phase 3 trial of vonicog alfa in a non-surgical setting were published in Blood in 2015.
The study included 49 patients with VWD who received vonicog alfa with or without recombinant factor VIII (FVIII).
All participants had successful treatment of bleeding episodes. Most (96.9%) treated bleeds (n=192 bleeds in 22 patients) were given an “excellent” efficacy rating (as good as or better than expected).
Most bleeds (81.8%) were resolved with a single infusion of vonicog alfa, and the treatment had a mean half-life of 21.9 hours.
There were 8 adverse events (AEs) considered related to vonicog alfa, and 2 were serious. One patient experienced 2 simultaneous serious AEs—chest discomfort and increased heart rate—but these were resolved.
There were no thrombotic events in this trial, no treatment-related binding or neutralizing antibodies against VWF, and no neutralizing antibodies against FVIII.
Surgical setting
Results from the phase 3 trial in a surgical setting were presented at the WFH 2018 World Congress.
The trial enrolled 15 adults with severe VWD who were undergoing elective surgical procedures (10 of them major procedures).
Patients received vonicog alfa at 40 to 60 IU per kg of body weight 12 to 24 hours before surgery. Within 3 hours of surgery, each patient’s FVIII level (FVIII:C) was assessed, with a target of 30 IU/dL for minor surgeries and 60 IU/dL for major surgeries.
Within an hour of surgery, patients received a dose of vonicog alfa, with or without recombinant factor VIII, depending on the target FVIII:C levels at the 3-hour assessment.
Ten patients received rVWF alone, 12 did not receive any preoperative FVIII, and 2 did not receive rVWF postoperatively.
The study’s primary endpoint was met. Vonicog alfa demonstrated overall hemostatic efficacy, as assessed 24 hours after the last perioperative infusion or the completion of the study visit, whichever occurred earlier.
Intra- and post-operative hemostasis was rated as “excellent” (as good as or better than expected) in 60% of patients and “good” (probably as good as expected) in 40% of patients.
One patient developed deep vein thrombosis 3 days after undergoing hip replacement surgery.
One patient tested positive for binding antibodies to VWF. None of the patients developed binding antibodies against potential impurities such as rFurin, CHO-protein, or mouse IgG.
The European Medicines Agency’s Committee for Medicinal Products for Human Use (CHMP) has recommended marketing authorization for vonicog alfa (Veyvondi), a recombinant von Willebrand factor (rVWF) product.
The CHMP is recommending vonicog alfa for the treatment of bleeding events and treatment/prevention of surgical bleeding in adults (age 18 and older) with von Willebrand disease (VWD) when desmopressin treatment alone is ineffective or not indicated.
The CHMP’s recommendation will be reviewed by the European Commission, which has the authority to approve medicines for use in the European Union, Norway, Iceland, and Liechtenstein.
The European Commission usually makes a decision within 67 days of the CHMP’s recommendation.
The CHMP’s opinion of vonicog alfa is supported by a pair of phase 3 trials.
Non-surgical setting
Results from a phase 3 trial of vonicog alfa in a non-surgical setting were published in Blood in 2015.
The study included 49 patients with VWD who received vonicog alfa with or without recombinant factor VIII (FVIII).
All participants had successful treatment of bleeding episodes. Most (96.9%) treated bleeds (n=192 bleeds in 22 patients) were given an “excellent” efficacy rating (as good as or better than expected).
Most bleeds (81.8%) were resolved with a single infusion of vonicog alfa, and the treatment had a mean half-life of 21.9 hours.
There were 8 adverse events (AEs) considered related to vonicog alfa, and 2 were serious. One patient experienced 2 simultaneous serious AEs—chest discomfort and increased heart rate—but these were resolved.
There were no thrombotic events in this trial, no treatment-related binding or neutralizing antibodies against VWF, and no neutralizing antibodies against FVIII.
Surgical setting
Results from the phase 3 trial in a surgical setting were presented at the WFH 2018 World Congress.
The trial enrolled 15 adults with severe VWD who were undergoing elective surgical procedures (10 of them major procedures).
Patients received vonicog alfa at 40 to 60 IU per kg of body weight 12 to 24 hours before surgery. Within 3 hours of surgery, each patient’s FVIII level (FVIII:C) was assessed, with a target of 30 IU/dL for minor surgeries and 60 IU/dL for major surgeries.
Within an hour of surgery, patients received a dose of vonicog alfa, with or without recombinant factor VIII, depending on the target FVIII:C levels at the 3-hour assessment.
Ten patients received rVWF alone, 12 did not receive any preoperative FVIII, and 2 did not receive rVWF postoperatively.
The study’s primary endpoint was met. Vonicog alfa demonstrated overall hemostatic efficacy, as assessed 24 hours after the last perioperative infusion or the completion of the study visit, whichever occurred earlier.
Intra- and post-operative hemostasis was rated as “excellent” (as good as or better than expected) in 60% of patients and “good” (probably as good as expected) in 40% of patients.
One patient developed deep vein thrombosis 3 days after undergoing hip replacement surgery.
One patient tested positive for binding antibodies to VWF. None of the patients developed binding antibodies against potential impurities such as rFurin, CHO-protein, or mouse IgG.
The European Medicines Agency’s Committee for Medicinal Products for Human Use (CHMP) has recommended marketing authorization for vonicog alfa (Veyvondi), a recombinant von Willebrand factor (rVWF) product.
The CHMP is recommending vonicog alfa for the treatment of bleeding events and treatment/prevention of surgical bleeding in adults (age 18 and older) with von Willebrand disease (VWD) when desmopressin treatment alone is ineffective or not indicated.
The CHMP’s recommendation will be reviewed by the European Commission, which has the authority to approve medicines for use in the European Union, Norway, Iceland, and Liechtenstein.
The European Commission usually makes a decision within 67 days of the CHMP’s recommendation.
The CHMP’s opinion of vonicog alfa is supported by a pair of phase 3 trials.
Non-surgical setting
Results from a phase 3 trial of vonicog alfa in a non-surgical setting were published in Blood in 2015.
The study included 49 patients with VWD who received vonicog alfa with or without recombinant factor VIII (FVIII).
All participants had successful treatment of bleeding episodes. Most (96.9%) treated bleeds (n=192 bleeds in 22 patients) were given an “excellent” efficacy rating (as good as or better than expected).
Most bleeds (81.8%) were resolved with a single infusion of vonicog alfa, and the treatment had a mean half-life of 21.9 hours.
There were 8 adverse events (AEs) considered related to vonicog alfa, and 2 were serious. One patient experienced 2 simultaneous serious AEs—chest discomfort and increased heart rate—but these were resolved.
There were no thrombotic events in this trial, no treatment-related binding or neutralizing antibodies against VWF, and no neutralizing antibodies against FVIII.
Surgical setting
Results from the phase 3 trial in a surgical setting were presented at the WFH 2018 World Congress.
The trial enrolled 15 adults with severe VWD who were undergoing elective surgical procedures (10 of them major procedures).
Patients received vonicog alfa at 40 to 60 IU per kg of body weight 12 to 24 hours before surgery. Within 3 hours of surgery, each patient’s FVIII level (FVIII:C) was assessed, with a target of 30 IU/dL for minor surgeries and 60 IU/dL for major surgeries.
Within an hour of surgery, patients received a dose of vonicog alfa, with or without recombinant factor VIII, depending on the target FVIII:C levels at the 3-hour assessment.
Ten patients received rVWF alone, 12 did not receive any preoperative FVIII, and 2 did not receive rVWF postoperatively.
The study’s primary endpoint was met. Vonicog alfa demonstrated overall hemostatic efficacy, as assessed 24 hours after the last perioperative infusion or the completion of the study visit, whichever occurred earlier.
Intra- and post-operative hemostasis was rated as “excellent” (as good as or better than expected) in 60% of patients and “good” (probably as good as expected) in 40% of patients.
One patient developed deep vein thrombosis 3 days after undergoing hip replacement surgery.
One patient tested positive for binding antibodies to VWF. None of the patients developed binding antibodies against potential impurities such as rFurin, CHO-protein, or mouse IgG.