Sergio López-Manzaneda1,2, Rebeca Sanchez-Dominguez1,2,Omaira Alberquilla1,2, Aida Garcia-Torralba1,2, Juan A. Bueren1,2, Oscar Quintana-Bustamante1,2, Susana Navarro1,2, Jose C Segovia1,2

1, División de Terapias Avanzadas en el Sistema Hematopoyético, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas - Centro de Investigación Biomédica en Red de Enfermedades Raras (CIEMAT/CIBERER). Madrid.

2, Unidad Mixta de Terapias Avanzadas. Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD). Madrid.

PYRUVATE KINASE DEFICIENCY AND THERAPEUTIC LENTIVIRAL VECTOR

Clinical Manifestations:• Chronic non-spherocytic haemolytic anemia

(CNSHA.)• Splenomegaly. • Reticulocytosis.• Fatal in some severely affected patients

during early childhood.

Energy Deficit

Hemolysis

We have designed a successful preclinical protocol in a PKDmouse model based on autologous transplantation ofhematopoietic stem cell (HSC) genetically corrected by aTherapeutic Lentiviral Vector (PGK-coPKLR.Wpre*-LV).

5’ pCCL

cPPTψ

PGK coRPK 3’ SIN

510 bp 1.7 Kb 610 pb

Wpre*

Pyruvate Kinase (PK),catalyzes the last step of Glycolysis

Loss in PK activity impairs the cell metabolism causingPyruvate Kinase Deficiency (PKD), an autosomalrecessive disease caused by mutations in the PKLR gene(Liver and Red Blood Cell isoforms.)

2 ADP

PK

C O

CH3

C

O O-

2C-O-PO3H2

CH2

C

O O-

2

Phosphoenolpyruvate Pyruvate

INTRODUCTION

RESULTS

CONCLUSIONS

AIMS

Define the minimal proportion of corrected cells required to achieve a therapeutic effect in PKD patients

EXPERIMENTAL DESIGN

Evaluation of phenotype correction VS wt engraftment

1st Approach: wt and PKD total bone marrow mixes

% wt cellsWt and PKD bone marrow cells mixed in different proportions

Cell mix transplant into conditioned PKD mice

515304060100 0

Evaluation of phenotype correction VS therapeutic vector copy number

502510310.30

Hematopoietic progenitors transplant into conditioned

PKD mice

MOIs (Multiplicity of infection) usedPKD hematopoietic progenitors transduced with different MOIs

2nd Approach: Transduction of PKD hematopoietic progenitors

Erythroid lineagefeatures oftransplanted mice withrespect to the wt/PKDproportion.Orange line, non-linealcorrelation. Dashedinterval in reticulocytesgraph representsstandard deviation ofcontrol non-transplanted PKD mice.Dotted horizontal linesare the thresholds setbased on ROC curvesperformed in controlnon-transplanted PKDmice.

Pink columnscorrespond to 15 to20% of wt cells amongall engrafted cells,interval which fits withthe minimumtherapeutic percentage.Mice transplanted withcell mixes containingless than 15-20% wtcells started to showhematological featuresthat fit with deficientmice.

Phenotype rescue depending on the wt/PKD cell proportion transplanted Phenotype rescue depending on therapeutic vector MOI used(A) Erythroid lineagefeatures of transplantedmice with different MOIof therapeutic vector.Orange line, non-linealcorrelation. Dashedinterval in reticulocytesgraph (standarddeviation non-transplanted PKD mice).Dotted horizontal linesare the thresholds setbased on ROC curvesperformed in non-transplanted PKD andnon deficient mice.Pink columnscorrespond to 0.2 and0.3 VCNs (vector copynumber), interval whichfits with the minimumtherapeutic VCN. Micethat showed VCN lower0.3 in peripheral bloodwere not capable toovercome the deficientphenotype.(B) Spleen size (left) andweight (right) atendpoint. Bothparameters arecorrelated with theVector Copy Number atend point

• PKD correction was found when total bone marrow contained at least 20% of non-deficient cells• PKD correction by the therapeutic vector was achieved when at least 0.3 copies of the vector were detected among the

total peripheral blood cell populations• Spleen Size and weight confirmed results obtained in peripheral blood and corroborated the therapeutic properties of the

developed clinical vector

PKD mouse model (AcB55)wt mice (C57BL/6)

Bone marrow cells extraction from wt and PKD mice

Transduction withTherapeutic

Vector

PKD mouse model (AcB55)

Bone marrow extraction

Hematopoietic progenitorssorting

Hematopoietic progenitors transduction

This vector have already being designated as Orphan Drug by theEuropean Medicines Agency (EMA) and Food and DrugAdministration (FDA).

FINDING THE MINIMUM VECTOR COPIES PER CELL NEEDED TO REACH PHENOTYPIC CORRECTION IN A MOUSE MODEL OF ERYTHROCYTE PYRUVATE

KINASE DEFICIENCY USING A CLINICALLY APPLICABLE LENTIVIRAL VECTOR

2ATP

A

B