Dr.ssa Costanza Lamperti

Gene therapy in MNGIE

Bologna 5-6/06/2015

Myo-Neuro-Gastro-Intestinal Encephalomyopathy

PHENOTYPE

PEO NeuropathyMyopathy

Leucoencephalopathy CachexiaInfections

MNGIE• Myo-neuro-gastro-intestinal

encephalomyopathy• Autosomal recessive• Combined mtDNA depletion, deletions,

mutations• Due to mutations in thymidine

phosphorylase

C DeplDelMult

Del

Why is mtDNA affected in MNGIE?

TP-Distribution

Distribution: Blood, Platlets,Spleen, Liver Lymphonode, Lung and Lymphocyte

Affected organs: Gastrointestinal, Skeletal Muscle,Peripheral Nerve and Brain

Therapy

Dialysis

Platelets and blood cells enzyme replacement

Liver transplant

AlloHSCT

Adeno Associated Virus (AAV)

• Parvovirus family;

• single strand DNA genome;

• small (25 nm);

• replication-defective (Dependovirus);

• nonenveloped.

Benefits: Long-term expression; Low immunogenicity; Wide tropism (serotype-dependent); Not currently known to cause disease; High genic transfer efficiency; Mitotic and post-mitotic infection; High titre production;

Disadvantages:o Transgene length (5 Kb);o Episomal;

AAV2 Serotypes

Ethylmalonic Encephalopathy:clinical, biochemical and genetics

Genetics Mutations in ETHE1

Vascular s. petechiae acrocyanosis

CNS early-onset hypotoniadevelopment delaylesions in brainstem

Gastrointestinal s. chronic diarrhoea

Biochemistry Cox deficiencyLactic acidosisEthylmalonic aciduriaelevated C4-C5 acylcarnitine high thiosulfatehigh H2S in tissues

Kootstra and Verma, 2003

AAV production

AAV2/8-TBG-hcTYMP

Horizon 2020

GT4MNGIE

September 2014: EMA

Phase I/II open-label dose escalation study

AAV INTRAVENOUS INJECTION

TBG-TYMP

dThd

dUrd

dThd dUrddThd

dUrd

dThddUrddThd

dUrd

dThddUrd

dThd

dUrd

dThddUrd

dThd

dUrd

dThddUrddThd

dUrd

dThddUrd

TP expressed in transduced hepatocytes

Effective systemic clearance of toxic

metabolites

Intravenous injection of vectorTYMP

Inclusion Criteria 1. Laboratory-confirmed MNGIE: thymidine phosphorylase (TP) defect (a and/or b): a) Homozygous or compound heterozygous mutations in the TYMP gene, and/or b) TP enzyme activity of <20% of normal,c) Increased plasma thymidine (Thd)> 3 micromole/L, ord) Increased plasma deoxyuridine (dUrd)> 7.5 micromole/L 2. Age at least 18 (18th birthday or later) on day that study consent is obtained. 3. Karnofsky performance status ≥50%/Lansky score>40%4. Agreement of investigators that AAV-TBG-TYMP is appropriate after discussion at Adult or Pediatric SCT Conference.8. Informed consent: Patients or their legal guardians must be aware of the nature of the disease process and must willingly give consent after being informed of the procedure, its experimental nature, alternatives, potential benefits, side-effects, risks, and discomforts.

Exclusion Criteria1. Severe cognitive impairment (e.g. I.Q. <70) that prevents the patient from participating in the treatment regimen reliably. 2. Severe psychiatric illness (i.e. depression, anxiety, etc. impairing work or activities of daily living) that interferes with the patient’s ability to comply with treatment regimen. 3. Presence of hepatitis (Positive HCV, HAV, HBV, or HBsAg).4. Moderate to severe hepatopathy (Liver cirrhosis on ultrasound. SGOT (AST) or SGPT (ALT) >2 x upper limit of normal (ULN) for age, total bilirubin ≥ 2x ULN unless the increase is attributable to Gilbert syndrome).7. Moderate to severe diabetes mellitus 8. Moderate to severe cardiomyopathy (in adults: congestive heart failure, ejection fraction <50%. In children: congestive heart failure, shortening fraction ≤27% by echocardiogram, or ejection fraction ≤50% by echocardiogram).9. Moderate to severe nephropathy (On dialysis or serum creatinine>2x ULN).10. For women of childbearing potential, participant cannot be pregnant, lactating, or plan to become pregnant over the course of the study. 11. Serious infection requiring antibiotics. 12. HIV disease (including HIV Ab positive).

 

Follow up• Timed water swallow • Questionnaires • DXA• Neurological exam • Nerve conduction study• Mini mental exam • Six minute walk test

UO Neurogenetica Molecolare Barbara GaravagliaValeria Tiranti Eleonora Lamantea Federica Invernizzi Silvia MarchetFranco CarraraIvano Di Meo

MRC CambridgeMassimo ZevianiCarlo ViscomiGabriele CivilettoRaffaele Cerutti

Manuela Bottani

Hospital Universitari Vall d’Hebron Research Institute (VHIR)

Ramon Marti

Javier Torres-Torronteras

Median= 72 days

Metronidazole+NACMetronidazole

Median=27 daysMedian= 55 days

METRONIDAZOLE, A BACTERICIDAL AGENT

Median=49 days

NAC

N-ACETYL CYSTEINE: A GSH PRECURSOR

*PRE

*POST

Ethe1-/-

mice

Ethe1-/-

children

AAV2/8-ETHE1 selectively infect the liver, restores SDO activity and increases lifespan of Ethe1-/- mice

Conclusions

AAV mediated gene therapy in liver is a realistic and feasible

approach;

AAV2/8 targets the liver in a very specific way;

AAV2/8 infection leads to robust expression of in liver, restoring the

TYMP activity, leading to a normalization of nucleotide pool in mice.

AAV mediated gene therapy is a promising approach for the

treatment for diseases due to accumulation of harmful compounds,

such as mitochondrial neurogastro-intestinal encephalomyopathy

(MNGIE), EE urea-cycle disorders or primary hyperoxaluria type I.

Do not need any immusoppression

H2S production: multiple sources

Exogenous: a by-product of anaerobic bacterial flora of the large intestine

Endogenous:a gasotransmitter”produced in trace by many organs

H2SGSSH

GSH SSO3

2-

H2S production: multiple sources

Exogenous: a by-product of anaerobic bacterial flora of the large intestine

Endogenous:a gasotransmitter”produced in trace by many organs

H2SGSSH

GSH SSO3

2-

ETHE1 -/- mouse model

• growth arrest from postnatal day 15

• reduced motor activity

• death around postnatal day 30

• COX deficiency in muscle, brain and colon

• high levels of ethylmalonic acid

• high levels of C4-C5 acylcarnitines

• high levels of thiosulfate and hydrogen sulfide (H2S)

-/- +/+

COX

acti v

it y ( %

)

**** **

Liver-targeted AAV-mediated gene replacement

• Parvovirus family;• single strand DNA genome;• small (25 nm); • replication-defective (Dependovirus);• nonenveloped.

Benefits: Long-term expression; Low immunogenicity; Wide tropism (serotype-dependent); Not currently known to cause disease; High genic transfer efficiency; Episomal

AAV2/8-TBG-hcTYMP

Why is mtDNA affected in MNGIE?