Flora Peyvandi

University of Milan

Terapia genica Cura per persone con emofilia?

Flora Peyvandi

Angelo Bianchi Bonomi hemophilia and Thrombosis Center

Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico

University of Milan, Italy

Bi-annual coagulation afternoon - IOSI - Istituto Oncologico della

Svizzera Italiana - Ospedale San Giovanni

08/10/2020 – Bellinzona

Flora Peyvandi

University of Milan

• Is a rare inherited bleeding disorder

• Is caused by deficiency or dysfunction of the coagulation protein FVIII

(hemophilia A) and FIX (hemophilia B) which have an essential role in

the coagulation cascade

• These dysfunctions cause a defect in clot formation and consequent

bleeding diathesis

Hemophilia

Physiol Rev. 2013;93:327-58.

Flora Peyvandi

University of Milan

The classification depends on the severity of the clotting factor deficiency

Classification Factor levelN° events

per yearPredisposition to bleeding

Severe < 1 IU/dL 24-48 Spontaneous/non-traumatic bleeding into joints or muscles

Moderate 1-5 IU/dL 4-6

Bleeding into joints and muscles after minor injuries or

after surgical intervention, occasional spontaneous

bleeding

Mild 5-40 IU/dL UncommonPost-operative and post-traumatic bleeding, spontaneous

bleeding is rare

Classification of hemophilia

Peyvandi F et al. Lancet. 2016;388:187-97; WFH - Guidelines for the management of hemophilia, 2nd 2012.

Flora Peyvandi

University of Milan

Spontaneous bleeding pattern

Stephensen et al. hemophilia 2009;15:1210-1214.

Children Adults

Joint 65% 52%

Muscle/soft tissue 23% 21%

Non-muscoloskeletal 12% 27%

Children AdultsAnkle 38% 60%

Knee 21% 12%

Hip 3.5% 4.3%

Wrist 2.6% 1.4%

Elbow 22.1% 14.3%

Shoul

der3.8% 4.3%

Finger 4.5% 2.9%

Flora Peyvandi

University of Milan

Synovial membrane

Cartilage

Recurrent haemorrhages in the same joint (ie, a target joint) cause:

1. Inflammation of the synovial tissue (ie, synovitis)

2. Progressive damage of the tissue

3. Development of arthropathy

Haemarthrosis vs arthropathy

Peyvandi F et al. Lancet. 2016;388:187-97.

Flora Peyvandi

University of Milan

• On-demand treatment is the infusion of the deficient clotting factor at the time of

bleeding

• Prophylactic treatment

- Regular infusions of the deficient clotting factor with the goal of preventingbleeding episodes

- Considered the standard of care in severe hemophilia

- Prophylactic regimen:

hemophilia A: 25-40 IU/kg 2-3 times per week

hemophilia B: 25-40 IU/kg 2 times per week

Treatment strategies

Manco-Johnson et al. NEJM 2007; Peyvandi et al. Lancet 2016.

• Is a replacement therapy based on the intravenous administration of the deficient

clotting factor

Flora Peyvandi

University of Milan

HIV

1950 1960 1970 1980 1990 2000 2010 2015 2020

HCV

Recombinant FVIII

and FIX concentrates

Viral-inactivated

concentrates

Plasma-derived

factor concentrates

Cryoprecipitate –Judith Graham Pool

Whole blood or

fresh plasma

F8 and F9

genes and cloning

Extended half-life FVIII and FIX

concentrates approved

Primary prophylaxis

became standard of care

History of available hemophilia therapies

Emicizumab approved by

FDA and EMA

Gene therapy in

HB and HA

Flora Peyvandi

University of Milan

Gene therapyReplacement therapy Non-replacement therapy

Standard

products

Extended

half-life

products

Palsma-derived

and recombinant

clotting factors

Anticorpo bispecifico

Anticorpi monoclonali

siRNA targeting antithrombin

First generation

• PEGylated

• Fc-fusion

• Albumin- fusion

Genetherapy

In-vivo

Ex vivoAnticorpi anti-TFPI

• Concizumab (Phase 3)

• Marstacimab (Phase 3)

• MG1113 (Phase 1)

Fitusiran (Phase 3)

Gene editing

Viral vector

Biengineered

recombinant

products

Pipe SW. Hematology Am Soc Hematol Educ Program. 2016;2016(1):650-656.

Srivastava A. WFH Guidelines for the Management of Hemophilia, 3rd ed. Haemophilia 2020. doi: 10.1111/hae.14046.

Hemophilia: Current and future approaches to care

SerpinPC (Phase1/2)

• Emicizumab• MIM8 (Phase 2)

• KY1049 (Preclinical study)

Second generation

• Intravenous

- rFVIIIFc-VWF-XTEN (BIVV001) (Phase 3)

• Subcutaneous

- SubQ8 (Phase1/2)

- HAT (Preclinical study)

Flora Peyvandi

University of Milan

The goal of gene therapy is to

replace the dysfunctional gene with

an exogenous functional gene

to cure the disease

Flora Peyvandi

University of Milan

Gene therapy In vivo

cDNA code for

therapeutic gene

(F8, F9)

Gene encapsulated in

AAVVector

administration

AAV releases

gene into cell Secreted protein

(FVIII o FIX)

Cellula target

Wang D, et al. Nat Rev Drug Discov. 2019;18:358-378.

Gene

expresses

proteins

Flora Peyvandi

University of Milan

Viral Vectors

Pros and Cons

• Viruses are used as vehicles to carry ‘functional' genes into human cells

Anguela XM and High K. Annu. Rev. Med. 2019. 70:273–88

Flora Peyvandi

University of Milan

Current status of the clinical trials in gene therapy

Flora Peyvandi

University of Milan

Gene Therapy Trials Throughout the World

Flora Peyvandi

University of Milan

Gene Therapy Trials

Anguela XM and High K. Annu. Rev. Med. 2019. 70:273–88

Hemophilia A and B

Flora Peyvandi

University of Milan

• Is becoming an exciting choice in the field of hemophilia treatment

• Clinical trials have demonstrated that one single intravenous

infusion of adeno-associated virus (AAV) vector containing F8 or

F9 cDNA can achieve:

– High protein expression levels

– Long-term durable factor expression

– Absence of spontaneous or traumatic bleeds

– Cessation of prophylaxis regimens

Gene therapy in hemophilia

Flora Peyvandi

University of Milan

AAV vectors

• Are considered the delivery tool of choice for in vivo therapy

• Non-pathogenic

• AAVs can transduce dividing and non-dividing cells

• A limitation of AAV vectors is the packaging size of the expression

cassette, approximately 5 kilobases (kb) of DNA

Peyvandi F, et al. Haemophilia. 2019;25(5):738-746

Flora Peyvandi

University of Milan

• Pre-existing immunity against AAV may limit the efficacy by

preventing liver transduction and therefore patient eligibility for such

treatment

• About 40% of the general population has a pre-existing immunity for

some AAV serotypes

• A geographic variability in seroprevalence of pre-existing immunity

against AAV serotypes has been recently reported

Pre-existing immunity

Hurlbut GD et al. Mol Ther. 2010; 18:1983–1994

Flora Peyvandi

University of Milan

Long-term expression

• AAV vector genome mainly persists as episome in the nucleus of

transduced cell

• Random integration events are

observed with a frequency of 10-4

- 10-5 with no preference for

specific genomic loci

Kaufmann KB et al. EMBO Mol Med 2013; 5:1642-1661; Colella P et al. Mol Ther 2018; 8:87-104

Flora Peyvandi

University of Milan

Overview of gene therapy in

hemophilia B

Flora Peyvandi

University of Milan

Gene therapy for hemophilia BClinical Trials 2003-2006

VectorRoute of

administrationResults

rAAV2 vector expressing hFIXIntramuscular:

skeletal muscle

- Effective gene transfer into muscle tissue

- Therapeutic levels of FIX were not seen

rAAV2 vector expressing hFIX Right hepatic artery

- Therapeutic levels of FIX attained for 2–4

weeks at the highest dose of vector

- Immune response against transduced

hepatocytes, abrogating clinical benefits

Manno CS et a. Blood. 2003; 101:2963–2972; Manno CS et al. Nat Med 2006; 12:342-347; Ray D et al. Expert Rev Hematol. 2011;4:539-49.

Flora Peyvandi

University of Milan

• Single dose of AAV8 vector infused in 6 patients

• FIX level at 2 to 11% in all patients

• FIX level was in a range of 1.4% to 7.21% over a

median period of 3.2 years in 6 patients

• FIX level was in a range of 2.8%‐6.6% in 4 newpatients

Clinical trial in hemophilia B

2010

2014

2018

• FIX activity level remained stable between 1.9%

and 5.1% over a period of at least 8 years with

a single dose of AAV8 vector

60th ASH Annual Meeting

Flora Peyvandi

University of Milan Nathwani AC et al NEJM 2014;371:1994-2004

• Self-limiting, capsid induced, immune mediated, asymptomatic

transaminitis occurred in 4/6 subjected treated at the high dose level but

controlled with low dose steroids without loss of transgene expression

Adverse events

Flora Peyvandi

University of Milan

Improvement of transgene expression

The gain-of-function mutation known as Padua (R338L)

increases FIX activity levels by 5 to 10 times compared with the

wild type FIX

Simioni P et al. N Engl J Med 2009;361:1671‐1675

Flora Peyvandi

University of Milan

Clinical trials in hemophilia B

Product name(s)Name(s) of active

ingredient

Developer /

manufacturer

Development

stage

AMT-061

etranacogene dezaparvovecPadua variant -AAV5 CSL Behring (UniQure)

Phase 3

SPK-9001

fidanacogene elaparvovecPadua variant - AAV-Spark100

Pfizer

(Spark)

FLT180a Padua variant - AAVS3 Freeline

Phase 1/2SB-FIX AAV6-delivered ZFN Sangamo

AMT-060 AAV5 UniQure

YUVA-GT-F901 HSC/MSC with lentivirus SGIMI Phase 1

TAK-748 (formerly SHP648/

AskBio009/BAX 335)

AAV8-based gene therapy using

FIX Padua variantTakeda Discontinued

Flora Peyvandi

University of Milan

SPK-9001

AMT-061

FLT-180a

AMT-060

scAAV278-LP1-hFIXco

Clinical trials in hemophilia B Results Phase 1/2

0

20

40

60

80

100

120

2x1011 6x1011 2x1012 5x1012 2x1013 5x1011 2x1013 4.5x1011 7.5x1011 9,75x1011

FIX

:C levello

(%)

Dose vettore (vg/Kg)

FIX Padua

7.3 7.9

22.9

41

37.5

1.9 2.3 5.1

31

98

>8 years

(St. Jude)

3 years

(UniQure)

>1 years

26 months

13

months

6,5 months

280253*

6,5 months

*Patient treated with anicoagulant drug

(Freeline)

CSL Behring

(UniQure)

Pfizer

(Spark)

1,5x1012

Flora Peyvandi

University of MilanWeek 0 time point reflects FIX activity before AMT-061 treatment; Week 0 and 2 samples may include activity from exogenous FIX replacement.

aPPT, activated partial thromboplastin time; FIX, factor IX.

• All participants achieved sustained mean FIX activity in the functionally curative

range after a single administration of AMT-061

50.2

31.3

40.8

0

10

20

30

40

50

60

70

0 4 8 12 16 20 24 28 32 36 40 44 48 52

FIX

activity o

ne-s

tage

aP

TT

(%

of n

orm

al)

Weeks following etranacogene dezaparvovec treatment

Participant 1 Participant 2 Participant 3

Mean FIX activity at 52 weeks: 41.0%

AMT-061: FIX Activity

Flora Peyvandi

University of Milan

aTransient, self-limiting headache, slightly elevated C-reactive protein; bHip surgery due to worsening of pre-existing condition avascular

necrosis; judged to be unrelated to treatment.

AEs, adverse events; ALT, alanine aminotransferase; AST, aspartate aminotransferase; FIX, factor IX; SAEs, serious adverse events;

TRAEs, treatment-related adverse events.

• AMT-060 and -061 were generally safe and well tolerated

AMT-060 5x1012 or 2x1013 gc/kg

(N=10)

AMT-0612x1013 gc/kg

(N=3)

TRAEs 4 in Cohort 1; 10 in Cohort 2

Most were mild and occurred in the first 3.5

months of follow-up

2 mild AEs possibly related to treatment in 1 participanta

SAEs No late-emergent SAEs 1 (judged to be unrelated to treatment)b

Liver enzymes Mild asymptomatic elevations in liver

enzymes in 3/10 patients 4- to 16-weeks

post-dosing

No loss of FIX activity

No clinically significant ALT or AST elevations

Immunosuppression 3 patients treated with corticosteroids None

FIX inhibitors None None

Safety

Flora Peyvandi

University of Milan

• Factor IX activity after 1 infusion of SPK-9001 in the 8 patients who did not

have an AAV-associated immune response

SPK-9001: FIX Activity

Flora Peyvandi

University of Milan

Overview of gene therapy in

hemophilia A

Flora Peyvandi

University of Milan

• Human FVIII coding sequence too large, 7 kb:

• Limited packaging capacity of AAV vector is 5,0 kb

• Expression of FVIII is highly inefficient

FVIIIViral vectorCodon optimize

The animal models showed an increase in FVIII

expression levels

Gene therapy in hemophilia APackaging problem

Flora Peyvandi

University of Milan

Clinical trials in hemophilia A

Product name(s) Name(s) of active ingredientDeveloper /

manufacturer

Development

stage

BMN-270

Valoctocogene roxaparvovecAAV5-huFVIII-SQ BioMarin

Phase 3SB-525

giroctocogene fitelparvovecrAAV2/6

Pfizer

(Sangamo)

SPK-8011 AAV-Spark200 Roche /Spark

BAY-19429/ BAY-2599023 AAVhu37FVIII Bayer Phase 1/2

Go-8AAV2/8-based

encoding FVIII-V3 variantUCL/St. Jude

Phase 1

ET3 HSC and lentivirus Expression Therapeutics

YUVA-GT-F801 HSC/MSC and lentivirus SGIMI

Pleightlet (MUT6)autologous CD34+PBSC, transduced with a

lentiviral vectorMC, Wisconsin

TAK-754 (formerly

BAX 888/SHP654)AAV8-based gene therapy (BDD)-FVIII-X5 variant Takeda Discontinued

Spark-8016 (Inhibs) AAV-Spark200 Spark Phase 1/2

AMT-180 (Inhibs)AAV5-based gene therapy using a FIX variant (FIX-

FIAV)UniQure Pre-clinical studies

Flora Peyvandi

University of Milan

0

10

20

30

40

50

60

70

2x1013 4x1013 6x1013 5x1011 1x1012 2x1012 2x1012 6x1012 3x1013 0,5x1013 1x1013

6 7

64,2

2

10

34

8

13

30

12,5

24

BMN-270

SPK-8011

AAV2/8-HPL-FVIII-V3

SB-525

BAY2599023

FV

III:C

le

ve

llo(%

)

Dose vettore (vg/Kg)

2 years

(Roche/Spark)

11 months

(UCL/St. Jude)

13 months

(Bayer)

15 months

Pfizer (Sangamo)

3-4 years

(BioMarin)

Clinical trials in hemophilia A

Results Phase1/2

Flora Peyvandi

University of Milan

BMN 270: FVIII ActivityFa

cto

r FV

III A

ctiv

ity

(IU

/dL;

Ch

rom

oge

nic

Ass

ay)

Study Week

N

24.2

7.99.9

16.4

6E13 vg/kg

4E13 vg/kg

Mean

Median

Flora Peyvandi

University of Milan

BMN 270: Safety profile of 6E13 and 4E13 cohorts

• The most common adverse events (AEs) included:

– Transient infusion-associated reactions

– Transient, asymptomatic, and mild to moderate ALT elevations with no long-lasting clinical sequelae

• No treatment-related serious adverse event (SAE) in the past year

• No clinical sequelae to corticosteroid use

• No thrombotic events

• No participants developed FVIII inhibitors

Pasi KJ et al. WFH Virtual Summit, 2020.

Flora Peyvandi

University of Milan

Human liver biopsy study following gene therapy for

hemophilia A

Fong S, et al. ISTH Virtual Congress, 2020.

• Examination of histopathology of the liver following BMN-270 therapy

• Quantification of FVIII DNA, RNA and protein expression in the liver to investigate inter-

individual variability

• Determination of vector DNA forms relating to durability of expression

Results

• Persisten FVIII expression was demonstrated

• The presence of circularised and ful-lenght hFVIII-SQ in human liver was detected,

nearly 4 years after a single AAV5-hFVIII-SQ infusion

Flora Peyvandi

University of Milan

SPK-8011: FVIII Activity

Flora Peyvandi

University of Milan

SPK-8011: Safety

• Follow-up (n = 14): Mean 27 months (range: 14-40); 31 patient-years

• No death, no FVIII inhibitor development

• Adverse events (AEs) related to SPK-8011:

– One participant experienced acute infusion reaction (presented as vomiting, pyrexia, back pain,

myalgia in the evening following completion of the vector infusion); all completely resolved

– Three participants reported LFT elevations. One of the events was Grade 2 transaminitis. All other

events were Grade 1. All events resolved

• SAE related to SPK-8011:

– One participant experienced Grade 2 transaminitis which resulted in elective hospitalization for IV

steroid administration, qualifying as an SAE. The event resolved

• AEs related to immunomodulatory agent–corticosteroids:

– Two participants experienced mild and non-serious steroid-associated AEs (eg weight gain, insomnia,

adrenal insufficiency, and worsening gastroesophageal reflux that resolved with medical intervention)

George L, et al. ISTH Virtual Congress, 2020.

Flora Peyvandi

University of Milan

Other gene therapy strategies

Flora Peyvandi

University of Milan

• Lentivirus is a class of retrovirus able to transduce dividing and

non-dividing cells

• Large transgene capacities: 7,5 Kb

• Advantage: the ability to integrate into the host genome via an

integrase allowing a long-term sustained transgene expression

• Lentivirus transduction can occur ex-vivo and in vivo

Lentiviral vector

Flora Peyvandi

University of Milan

Preclinical study with lentiviral vectors

• Newborn mice treated with lentiviral vectors (LV) showed that transduced hepatocytes

are maintained during growth and proliferate locally

• Administration of LV in 2-weeks old mice resulted in stable and 3-fold higher FIX output

compared to mice treated as neonates

• These data support further progress of gene therapy with LV; potentially extending its

accessibility to pediatric patients

Milani M L, et al. ISTH Virtual Congress, 2020.

Flora Peyvandi

University of Milan

• Genome editing approaches are a simple and robust technology to

modify the genome of living cells

• Nucleases used include:

- Zinc-finger nucleases (ZFN)

- Clustered regularly interspaced short palindromic repeat (CRISPR)

- CRISPR associated 9 (Cas9) nucleases

Genome editing

Dunbar et al. Science 2018; 359: eaan4672; Adli M et al. Nat Commun 2018;9:1911

Flora Peyvandi

University of Milan

• The increase in liver enzymes

• The safety profile of different AAV serotypes

• The effect of the vector manufacturing process

• The potential genotoxicity derived from integrating gene delivery

vectors

• The discrepancy in FVIII and FIX activity assays

Critical issues

Flora Peyvandi

University of Milan

Liver enzymes

• Elevation in liver transaminase is the main toxicity observed

• The majority of events have been managed with corticosteroids

• Some cases have been associated with partial or complete loss of

transgene expression

• The pathophysiological mechanism for the liver toxicity remains

unclear

• A capsid-specific cytotoxic T cell responses against the vector-

transduced cells could be an explanation of this liver toxicity

Mingozzi F et al. Nat Med. 2007;13:419–422; Ertl HCJ et al. Hum Gene Ther. 2017;28:328-337

Flora Peyvandi

University of Milan

Assays

• Discrepancy in FVIII and FIX activity assays measured by the

standard one-stage clotting assays and chromogenic assays

• These discrepancies were observed in haemophilia A patients who

received an AAV vector expressing a B-domain deleted FVIII

transgene, and in haemophilia B patients who were infused with an

AAV vector carrying F9 cDNA with Padua variant

Rangarajan S et al. N Engl J Med. 2017;377:2519-2530; Robinson et al. ASH Meeting 2018; Lengler J et al. ISTH Congress 2019

Flora Peyvandi

University of Milan

Safety and surveillance

Flora Peyvandi

University of Milan

• Patients in gene therapy study may be at increased risk of undesirable and

unpredictable outcomes which may present as delayed ADEs

- Risk of tumorigenicity

- Unwanted immunogenicity and its consequences

- Risks related to infection with vectors used in gene therapy

- Risk of insertional oncogenesis

• The design of long term follow-up observational studies are required for

the collection of data on delayed ADEs following administration of a gene

therapy product also in the post-marketing phase

Gene therapy

Long term follow-up

Flora Peyvandi

University of Milan

Long Term Follow-Up After Administration of Human Gene Therapy Products – FDA July 2018

Gene therapy: FDA and EMA recommendations

• 15 years for integrating vectors such as gammaretroviral and

lentiviral vectors and transposon elements

• Up to 5 years for AAV vectors

• Up to 15 years for genome editing products

Guideline on safety and efficacy follow-up and risk management of Advanced Therapy Medicinal Products– EMA Jannuary 2018

• For gene therapy medicinal products using integrating vectors or

have the potential for latency followed by reactivation, it is

usually expected to follow the patients up to 15 years

FDA

EMA

Flora Peyvandi

University of Milan

• The aim is to provide a robust, scientifically valid data collection avenue,

available to all healthcare providers treating patients who receive gene

therapy

Flora Peyvandi

University of Milan

• The FDA recommended BioMarin complete the Phase III trial and submit

two-year follow-up safety and efficacy data on 134 patients

• In addition, the FDA introduced a new recommendation for two years of

data to provide substantial evidence of a durable effect using Annualized

Bleeding Rate (ABR) as the primary endpoint

FDA rejects BioMarin’s gene therapy for hemophilia

Flora Peyvandi

University of Milan

• Unknown, but based on 2 year data request:

– 56% drop in FVIII activity between end of year 1 and end of year 2 in Phase 1/2 trial

– Some patients in Phase 3 had no response to the vector

– Phase 3 26-week results are lower than results seen in Phase 1/2 at same dose and

time point

– Data from full study at 2 years would provide more information

• Spark-Roche released data on AAV-LKO3-SQ-FVIII at ISTH 2020

– High variability in 7 high dose patients; 2/7 lost all FVIII activity presumed due to

cytotoxic T-cell response not controlled by steroids

Why Did FDA Reject BioMarin Application?

Flora Peyvandi

University of Milan

- Gene therapy has been one of the biotech’s biggest successstories of the 21st century

- A promising modality for future medicine applicable to a broadnumber of diseases

- More information is needed to understand the safety of thisinnovative therapy

Considerations

Flora Peyvandi

University of Milan

Clinical

Delivery

Access

Site

Preparedness

Safety/EfficacyEducation

C

i

Pathway to Preparedness