“Nuoviapproccidiricercae’ terapiagenica ...€¦ · •In vivo versus ex vivo • In vivo =...
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“Nuovi approcci di ricerca e terapia genica, il coinvolgimento dell’Italia: a che punto siamo?” Convegno Annuale AICE ASSISTENZA DELL’EMOFILIA E DELLE MEC IN ITALIA: SCENARI IN EVOLUZIONE. Napoli, 30 se-embre • 2 o-obre 2015 Flora Peyvandi Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico and University of Milan, Italy
Transcript of “Nuoviapproccidiricercae’ terapiagenica ...€¦ · •In vivo versus ex vivo • In vivo =...
“Nuovi approcci di ricerca e terapia genica,
il coinvolgimento dell’Italia: a che punto siamo?”
Convegno Annuale AICE ASSISTENZA DELL’EMOFILIA E DELLE MEC IN ITALIA:
SCENARI IN EVOLUZIONE. Napoli, 30 se-embre • 2 o-obre 2015
Flora Peyvandi Angelo Bianchi Bonomi Hemophilia and Thrombosis Center,
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico and University of Milan, Italy
• Genes, the functional unit of heredity, encode instructions to make proteins
• When genes are altered, encoded proteins are unable to carry out their normal functions, resulting in genetic disorders
• Gene therapy is basically to replace the dysfunctional gene with an exogenous functional gene to cure the disease phenotype
Goal of Gene therapy
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• In vivo versus ex vivo • In vivo = delivery of genes takes place in the body • Ex vivo = delivery takes place out of the body, and then cells
are placed back into the body
Different Delivery Systems
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(Turitz Cox et al. Nature Medicine 2015;21:121–131)
• In vivo versus ex vivo • In vivo = intravenous or intramuscular or non-invasive (‘sniffable’) • Ex vivo = hepatocytes, skin fibroblasts, haematopoietic cells
(‘bioreactors’)
• Gene delivery approaches • Physical methods • Non-viral vectors • Viral vectors
GeZng genes into cells
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• Viral – Integrated into DNA
• Retrovirus (RNA-‐based) • Len\virus (RNA-‐based)
– Adeno-‐associated (DNA-‐based)
– Non-‐integrated • Adenovirus (DNA-‐based) • Herpesvirus (DNA-‐based)
• Non viral
– Liposomes – Lipocomplexes – Naked plasmid DNA – Receptor-‐ mediated transfer
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Types of gene therapy Gene delivery systems
Genome integration Insert size Duration of
expression
No 45 kb Transient
No ∼5,0 kb Long-lasting???
Yes 7-10 kb Long-lasting
Yes 9-10 kb Long-lasting
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Viral vectors -‐ Pro vs cons
What takes to get a successful gene therapy
Being able to infect the target
cells Being able to induce a sustained transfection
Being able to induce a
sustained clinical response
Being able to manufacture a loaded vector
Being able to reach the target cells
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Gene therapy
• In 1990, the first successful clinical trial of gene therapy was ini\ated for adenosine deaminase deficiency
(Blaese et al. Science 1995; 270:475-‐80)
• In 1984, retroviral transfer of gene\c material into hematopoie\c stem and progenitor cells led to predic\ons that this technology would transform modern medicine
• In 2012,1843 trials undertaken in 31 countries
(Ginn et al J Gene Med 2013; 15:65-‐77)
Why gene therapy for haemophilia?
• Current treatment with plasma-‐ or recombinant products - safe and highly effec\ve but not cura\ve, invasive, and demanding
• Gene therapy offers the poten\al for a cure
Haemophilia is well suited for gene therapy approaches
• Single gene defect (FIX or FVIII gene) • Therapeu\c goal modest
- an increase in plasma FIX/FVIII levels above 1% would be sufficient ameliorate the bleeding phenotype
• Efficacy can be assessed by validated rou\ne laboratory assay
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Viral vector Number of paIents Method Safety DuraIon of
expression
Expression of transgene product
Status Reference
FVIII
Non-‐viral vector 6
Dermal fibroblasts transfected with plasmid containing FVIII
Transient expression ~6 months 0,8 – 4% Closed
(Roth et al, NEJM 2001)
Moloney murine leukemia virus – MLV (retroviral vector) BDD, in vivo
13 Peripheral vein infusion
Detec\on of vector genome in semen
~1 years 1 – 2% Trial terminated
(Powell et al, Blood 2003)
Adenoviral vector, full length human FVIII, in vivo 1 Intravenous
infusion
Increase AST/ALT, thrombocytopenia and fever, immuno reacIon to the virus
Transient 3% Closed
GenStar-‐Therapeu\cs, 2003 (Data not published)
FIX
rAAV-‐FIX (serotype 2), in vivo 8 Intramuscular injec\ons
No significant side-‐effects Transient <1,6% Closed (Manno et al,
Blood 2003)
AAV2-‐FIX, in vivo 7 Hepa\c artery infusion
Detec\on of vector genome in semen of subjects and increase ALT
~8 weeks 3 – 11% Sospended
(Manno et al, Nature Medicine 2006)
Clinical trials – Haemophilia A and B
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Discussion on clinical trials
• Several ques\ons and issues remain:
– The FVIII and FIX plasma levels reached are insufficient to free pa\ents from the need of exogenous factors infusion
– Expression of the transgene has been rela\vely short-‐las\ng
– The poten\al for inhibitor development is s\ll of concern
– The risk of germline integra\on and passage of the vector to descendants cannot be ruled out
– Immunotoxicity, immunity to AAV that blocks vector delivery and precludes efficient liver transduc\on
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Discussion on clinical trials
• Several ques\on and issues remain:
– The FVIII and FIX plasma levels reached are insufficient to free pa\ents from the need of exogenous factors infusion
– Expression of the transgene has been rela\vely short-‐las\ng
– The poten\al for inhibitor development is s\ll of concern
– The risk of germline integra\on and passage of the vector to descendants cannot be ruled out
– Immunotoxicity, immunity to AAV that blocks vector delivery and precludes efficient liver transduc\on
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Poten\al solu\on
• Strategies to overcome prevalent immunity to AAV:
– Immunosuppression at the \me of vector administra\on
– Different AAV serotypes
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Alternate serotypes of AAV
• Early clinical trials using an AAV serotype 2 elicited immune response, leading to loss of expression
• Several clinical trials tes\ng different AAV vectors have guided the understanding towards development of op\mal configura\on of an adequate gene therapy approach
• It is of importance to limit any effects of possible pre-‐exis\ng immunity to AAV
• Prevalence of an\bodies against to AAV:
– AAV5 (3,2%) – AAV8 (19%) – AAV2 and AAV1 (59% and 50,5% respec\vely)
To circumvent any effects of possible pre-‐exisIng immunity, AAV8 and AAV5 were chosen
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• Developed an AAV vector serotype 8 capsid with low seroprevalence in humans
• Therapeu\cally relevant levels of transduc\on could be achieved with intravenous infusion
Gene therapy in haemophilia B Trial in nonhuman primate
• Peripheral vein infusion of 1x1012 vg/kg scAAV-‐LP1-‐hFIXco pseudotyped with serotype 8 capsid, in 3 macaques, resulted in stable therapeu\c expression (more than 9 months) of human FIX (hFIX)
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The most recently published clinical trial demonstrated that sufficient FIX expression can be achieved through intravenous infusion of AAV8-‐hFIX in haemophilia B pa\ents thus conver\ng severe haemophilia B phenotypes to moderate/mild haemophilia B.
(Nathwani A et al, N Engl J Med 2011;365:2357-2365 )
Haemophilia B gene therapy in human
Haemophilia B gene therapy with self complementary AAV vector
• Study opened in March 2010
• 10 paIents recruited to date: - 2 with low (2x1011vg/kg) dose - 2 with intermediate (6x1011vg/kg) dose - 6 with high (2x1012vg/kg) dose
Flora Peyvandi (Nathwani A et al, N Engl J Med 2011;365:2357-2365 )
(Presented by Dr Amit C. Nathwani, UCL/St Jude Childrens Research Hospital at WFH 2014)
Haemophilia B gene therapy trials Results
• Human FIX expression between 1 -‐ 6% in all 10 subjects treated to date with follow-‐up between 0.8-‐3.5 years
- 5/7 able to stop prophylaxis, with significant improvement in quality of life and no spontaneous bleeds
- 4/6 high dose pa\ents have not required any treatment following gene transfer
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• Self-‐l imi\ng, capsid induced, immune mediated, asymptoma\c transamini\s occurred in 4/6 subjected treated at the high dose level but controlled with low dose steroids without loss of transgene expression
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Current status of haemophilia B gene therapy
Responsible Viral vector SomministraIon Status Number of Clinical trial
St. Jude Children's Research Hospital
Self Complementary Adeno-‐Associated Viral Vector (scAAV 2/8-‐LP1-‐hFIXco)
Peripheral vein infusion Phase I (is currenly recrui\ng) NCT00979238
Spark Therapeu\cs Single-‐Stranded, Adeno-‐Associated
Pseudotype 8 Viral Vector (AAV8-‐hFIX19 vector)
Intravenous dose Phase I/II
(is ongoing, but not recrui\ng par\cipants)
NCT01620801
Spark Therapeu\cs Adeno-‐Associated Viral Vectors (AAV2-‐hFIX16)
Intrahepa\c administra\on
Phase I (is ongoing, but not
recrui\ng par\cipants) NCT00515710
Spark Therapeu\cs Novel recombinant adeno-‐associated virus (AAV) (SPK-‐9001) Intravenous dose
Phase I/II (is not yet open for
par\cipant recruitment) NCT02484092
Baxalta US Inc. Self-‐Complemen\ng Op\mized
Adeno-‐associated Virus Serotype 8 (BAX 335)
Intravenous dose Phase I/II (is currenly recrui\ng) NCT01687608
UniQure Biopharma B.V.
Adeno-‐associated Viral Vector Containing a Codon-‐op\mized Human Factor IX Gene (AAV5-‐
hFIX)
Intravenous dose Phase I/II (is currenly recrui\ng) NCT02396342
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• A Phase I/II open-‐label, uncontrolled, single dose, dose ascending, mul\-‐centre trial inves\ga\on an adeno-‐associated viral vector containing a codon-‐op\mized human FIX (hFIX) gene administered to adult pa\ents with severe or moderately severe haemophilia B
• Primary objecIve of is to inves\gate the safety of systemic administra\on of AAV5-‐hFIX to adult pa\ents with severe or moderately severe haemophilia B
• A number of secondary objecIves will be addressing the efficacy and safety of systemic administra\on of AAV5-‐hFIX
Haemophilia center of Milan & UniQure
• Subjects will be allocated to one of two cohorts with the following planned dose levels: -‐ Cohort 1 (5 subjects): AAV5-‐hFIX 5.0 × 1012 gc/kg -‐ Cohort 2 (5 subjects): AAV5-‐hFIX 2.0 × 1013 gc/kg
• The cohorts will start sequen\ally and dosing of first, second and third subject within a cohort will be separated by an interim review of the safety data
• Cohort 2 will be ini\ated based on the decision of the safety commivee awer review of safety data from Cohort 1. These data will include 12-‐week follow-‐up for the first 3 subjects dosed as well as four weeks follow-‐up for the remaining 2 subjects in cohort 1 -‐ If liver enzyme perturba\ons requiring cor\costeroid treatment in one or more of the first three subjects in Cohort 1, the safety commivee may decide to extend the follow-‐up period, before a decision to ini\ate Cohort 2 is taken.
Trial cohorts
Clinical Trial Period -‐ First Subject Screened (Es\mated): Q4-‐2014 -‐ Last Subject Last Visit (Es\mated): Q4-‐2020 Visits During the en\re course of the study, there will be at total of 35 scheduled visits for each pa\ent at the clinic: -‐ 1 screening visit -‐ 1 dosing visit -‐ 19 visits up to week 26 following AAV5-‐hFIX infusion -‐ 10 visits from week 26 to year 3 -‐ 4 visits from 3 to 5 years following AAV5-‐hFIX infusion. ReporIng of bleeds and treatment with FIX Throughout the study period, pa\ents will record the use of FIX replacement therapy and informa\on on bleeding episodes in a web based electronic diary (e-‐diary)
Clinical trial period and visits
Primary endpoint -‐ Adverse Events Secondary endpoints -‐ Efficacy
• FIX-‐replacement-‐therapy-‐free FIX ac\vity • Bleeding rate • Total consump\on of FIX replacement therapy • Quality of life using the SF-‐36 Safety
-‐ Safety
• Vector DNA in body fluids • Neutralising an\bodies to AAV5 • Total IgM and IgG an\bodies to AAV5 • AAV5 capsid-‐specific T cells • An\bodies to FIX • FIX inhibitors • Inflammatory markers: IL-‐1β, IL-‐2, IL-‐6, INFγ, MCP-‐1
Endpoints
Haemophilia A gene therapy
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Limita\ons in haemophilia A gene therapy
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• Expression of FVIII is highly inefficient
• Packaging capacity of AAV vector - Limited packaging capacity of AAV is ~5.0 kb
- Large transgenes, such as FVIII (7.0 kb), complicate gene therapy applica\ons using viral vector, compromising vector \ter and efficacy
• Codon opImized constructs achieved a 29-‐ to 44-‐fold increase in expression compared with expression from equivalent non–codon-‐op\mized sequences (P<0.0001, Bonferroni simultaneous test)
• Shorter FVIII constructs can be more easily accommodated in viral vectors can result in increased therapeu\c efficacy
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Expression of FVIII
Challenges to haemophilia A gene therapy
• A codon-‐opImized hFVIII cDNA: 226 amino acid B-‐domain spacer replaced the en\re B domain and a hybrid liver-‐specific promoter (HLP) mediated 10-‐fold higher hFVIII levels in mice compared with non–codon-‐op\mized variants
• A further twofold improvement in potency was achieved by replacing the 226-‐aa N6 spacer with a novel 17-‐aa pepIde (V3)
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Challenges to haemophilia A gene therapy
• rAAV-‐HPL-‐codon-‐hFVIII-‐V3 mediated higher hFVIII an\gen expression in two different mice models
• Stable hFVIII expression was observed in non-‐human primate
Flora Peyvandi (McIntosh J et al, Blood. 2013;121:3335-3344)
• hFVIII expression above 100% was observed in 3 macaques but these animals developed neutralizing an\-‐FVIII an\bodies
• Bioengineering of the FVIII has resulted in a potent pep\de HLP-‐codop-‐hFVIII-‐V3 expression casseve that has improved the potency and packaging efficacy into rAAV vectors
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• Limited packaging capacity of AAV was bypassed using two different vectors, one for the heavy chain and one for the light chain of FVIII
The packaging capacity
• The two vector plasmids get cotransfected into the host produc\on cells at the same \me.
• Expression level of FVIII obtained cotransfected dual-‐vector into 293 cells and in HA mice were similar to standard vector.
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• Len\viral vectors are able to transduce both dividing and non-‐dividing cells1, but unlike adeno-‐associated viral vectors are not as constrained by the size of the transgene.
• Len\viral vectors have a carrying capacity of approximately 9-‐10kb exogenous DNA
• Third genera\on len\viral vectors have been used for gene therapy applica\ons without any adverse complica\ons2
• No direct evidence for inserIonal mutagenesis has been documented for recombinant len\viral-‐based retroviruses
(1Naldini et al, Proc Natl Acad Sci U S A. 1996;93:11382-‐8) (2Car\er et al, Science. 2009;326:818-‐23.)
The packaging capacity LenBviral vectors
• gene therapy is emerging as a powerful and viable way to treat haemophilia B and encouraging results have been achieved in pa\ents
-‐ a stable expression of FIX has been achieved through intravenous infusion of AVV8-‐hFIX in HB pa\ents conver\ng the severe phenotypes to moderate/mild (Nathwani et al, 2011)
• despite many efforts in haemophilia A gene therapy, this therapeu\c target is more problema\c for a variety of reasons:
- the larger factor VIII cDNA
- achieve adequate levels of transgene expression
- complica\on of an\factor VIII immunity
Considera\ons
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BioMarin Enrolls First Pa\ent in Phase ½ with BMN 270 for the treatment of Hemophilia A-‐ AAV
Breaking News!
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