pPLUS® AAV-Helper, novel engineered pHelper plasmid to improve yieldand quality of several AAV serotypes in suspension cell culture systems.
Eric Mauro2, Jonathan Havard1, Laure Robert1, Alengo Nyamay’antu1, Carine Morel2, Julie Regnery3, Elisa Berera3, Samantha Convers3, Pauline Schorter3, Quentin Bazot3, Sylvain Julien2, PatrickErbacher1
1 Polyplus®, Illkirch, France 2Polyplus®, Loos, France 3ABL, Lyon, France
Abstract

Harnessing rAAVs as viral vectors for therapeutic transgene delivery still requires improvements in yields and specificity to lower vector doses, and therefore manufacturing cost, as well as to improve patientsafety. To this end, our research is focused on developing novel technologies to ensure manufacturing of high yielding rAAV particles using transient transfection, as well as enhancing features of rAAV vectorsthat act on the overall size of packaged material and specificity of delivery. Here we present our state-of-the art approach to design new helper plasmids (phelpers) with the aim of improving both theinfectiosity (TU/mL) and the quality (full/empty ratio) of the viral particle obtained from suspension cultures. We took the opportunity to exploit our proprietary DNA assembly method technology to explore
the synergies of multiple genetic features modularly assembled in synthetic plasmids. Comparison of the biological activity of several versions of rationally designed pHelpers led us to identify the optimal
configuration able to outperform existing helper plasmids in every tested bioproduction conditions. Our expertise in DNA plasmid design and assembly together with our scalable transfection solutions forrAAV manufacturing gives us the potential to improve both productivity and specificity of gene therapy products.

Introduction: rAAV production

StrategyTriple transfection method
Various constructions of pHelper were madeusing e-Zyvec® plasmid assembly technology.
Plasmids were transfected into HEK-293T cells
and harvested 72h post-transfection.

pHelper, pRepCap and pTransgene
are transfected into HEK-293T cells

72h post-transfection, cells are lysedand viruses harvested

Removing non essential elements

Viral titer is quantified by flow cytometry

Shuffling

Construction of a minimal pHelper (mpH).

Addition of new Helper elements

Re-organization of plasmid configuration to optimize its
efficiency to produce rAAV.

Fig1: Viral titers of rAAV2 (normalized to pALD-X80). Copy/cellof the pHelpers are shown above plasmids name.
19 constructions tested, 3 series

Viral titers (TU/mL) of the produced rAAV werethen compared to identify the best pHelper for
optimal rAAV production.

Identification of new sequences to add to the pHelper to boost
rAAV production.

Fig2: Viral titers of rAAV2 (normalized to pALD-X80). Copy/cellof the pHelpers are shown above plasmids name.
9 constructions tested, 2 series

Fig3: Viral titers of rAAV2 (normalized to pALD-X80). Copy/cellof the pHelpers are shown above plasmids name.
29 constructions tested, 5 series

pPLUS® AAV-Helper to improve yield and quality of different AAV serotypesA)

Optimized titers when using FectoVIR®-AAV in combination with pPLUS® AAV-HelperB)

9Improved titers with pPLUS® AAV-Helper for different serotypes
383

6

Fold change

Fold change

754322121

10
pALD-HELP

pPLUS® AAV-Helper

pALD-HELP

Competitor transfection reagent

pPLUS® AAV-Helper

pALD-HELPPEIpro®

pPLUS® AAV-Helper

FectoVIR®-AAV0
VG

TU
AAV2

VG/mL meanC)

TU/mL Fold increase

VG
AAV5
pALD-HELP

TU

VG

AAV8
pPLUS® AAV-Helper

TU

VG

TU
AAV9Improved full/empty particle ratio with pPLUS® AAV-Helper

Fig 4: pPLUS® AAV-Helper is a novel helper plasmid for AAV production in suspension HEK-293 cell lines to improve
production yields of all AAV serotypes tested (AAV2, AAV5, AAV8, AAV9).
A) Improved productivity and infectivity with an optimized efficiency when used in synergy with FectoVIR®-AAVtransfection reagent. AAV9 were produced in HEK293T cells adapted in suspension in F17 medium and transfected withFectoVIR®-AAV. Transduction assay was performed on HEK293T cells.
B) Improved titers with pPLUS® AAV-Helper for different serotypes. AAV2, AAV5, AAV8 and AAV9 were produced inHEK293T cells adapted in suspension in F17 medium and transfected with FectoVIR®-AAV.Transduction assay wasperformed on HT-1080 cells. Improvement of titers are shown as fold increase in reference to pALD-HELP.
C) Improved full/empty particle ratio with pPLUS® AAV-Helper. AAV2 was produced in HEK293 suspension cells andtransfected with FectoVIR®-AAV. Two idependents experiments were conducted where each transfection wasperformed in duplicate. Three different plasmids ratio were evaluated (Transgene; Helper; RepCap). The % of full capsids
showed is the results of qPCR:ELISA ratio (data provided by ABL).