An alternative to viral vectors for Gene and Cell Therapies,in vivo-jetPEI®

Valérie Kedinger, Pascale Belguise, Anne-Laure Bellemin, Marie-Elise Bonnet, Géraldine Guérin-Peyrou, Patrick Erbacher

Polyplus-transfection, Bioparc, 850 Boulevard S. Brant, 67400 Illkirch, France

AbstractAdvanced therapy medicinal products (ATMPs) including gene and cell therapy medicines have emerged aspromising treatments for various diseases. These therapies involve the introduction of a therapeutic nucleic acidinto the patient’s body or patient’s cells. in vivo-jetPEI®, a cationic polymer-based reagent, is a very powerfulnon-viral vector to safely and easily deliver nucleic acids in vivo to a wide range of tissues through various routesof administrations. in vivo-jetPEI® offers high performance in terms of efficiency, reproducibility and robustness.Following in vivo-jetPEI®-mediated systemic delivery of nucleic acid, no induction of major pro-inflammatorycytokines and no increase in sera levels of hepatic enzymes is observed, making it a reliable and safe alternativeto viral vectors that can elicit an immune response. Nowadays, in vivo-jetPEI® is a widely used chemical reagentto deliver nucleic acids in animals and it has been selected as the delivery vector of choice in several drugdevelopment programs. To fulfill all the quality requirements associated to its use in Human, Polyplus-transfection® can supply preclinical grade as well as cGMP grade in vivo-jetPEI® reagents that are usedworldwide for a growing number of preclinical studies and clinical trials based on plasmids or oligonucleotidesdelivery.

Very easy to handle protocol

High stability of complexes formed betweennucleic acid and in vivo-jetPEI®

Complexes were formed at 200 µg/ml of siRNA and at an N/P of 8. Complexes size was measured by Dynamic Light Scatteringand charge was measured using a zeta sizer after 1, 3, 7, 14, 21 and 28 days of incubation at 4°C (Left panel). After differenttimes of incubation at 4°C, complexes were analyzed by gel electrophoresis with (Right panel, bottom part) or without SDStreatment to monitor siRNA integrity (Right panel, top part).

Complexes protect integrity of nucleic acids in presence of serum

Size and charge of complexes over time

Nucleic acid/in vivo-jetPEI® complexes are very stable over time

• Manufacturing of in vivo-jetPEI® in compliance with US and EU GMP guidelines since2007.

• Several clinical trials worldwide using in vivo-jetPEI® for the delivery in human of differenttypes of nucleic acids (DNA, siRNA, oligonucleotides…) (Buscail et al., 2015; Sidi et al.,2008; Matouk et al., 2013).

• Different administration routes can be used, including systemic delivery• Used in different applications such as in cancer therapy, immunization, modulation of

blood-brain barrier.• in vivo-jetPEI® mediated delivery of nucleic acids can be used as a treatment in

combination with chemotherapy.• One project entering Phase III in 2018.

Complexes were formed at a constant N/P ratio of 8 and incubated for 4 hours at room temperature either in 5% glucose withoutserum or in 5% glucose with 50% of serum. After incubation, complexes were analyzed by gel electrophoresis (Left part), or treatedwith SDS to release siRNA from the complexes and then analyzed by gel electrophoresis (Right part).

Complexes were formed in 200 µl of 5% glucose using 40 µg of luciferase expressing plasmid with in vivo-jetPEI® at an N/P ratio of8, and injected through retro-orbital sinus. 24 hours after injection, blood was collected and the level of LDH, ASAT, ALAT and ALPwas measured. Each value corresponds to the mean ± SD (n=8). As a positive control, CCl4 was subcutaneously administered.

No induction of liver enzyme

Safe method of delivery, with no major inflammatory response triggered

No pro-inflammatory cytokine expression

Complexes were formed in 200 µl of 5% glucose using 40 µg of luciferase siRNA with in vivo-jetPEI® at an N/P ratio of 8, andinjected through retro-orbital sinus. 1 to 6 hours after injection, blood was collected and the level of TNF, IFN and IL-6 wasmeasured by ELISA (n=8). As a positive control, LPS was injected intraperitoneally.

www.polyplus-transfection.com

Bonnet, M.E., P. Erbacher, and A.L. Bolcato-Bellemin. 2008. Pharmaceutical research. 25:2972-2982.Bonnet, M.E., J.B. Gossart, E. Benoit, M. Messmer, O. Zounib, V. Moreau, J.P. Behr, N. Lenne-Samuel, V. Kedinger, A. Meulle, P. Erbacher, and A.L.Bolcato-Bellemin. 2013. Journal of controlled release. 170:183-190.Buscail, L., B. Bournet, F. Vernejoul, G. Cambois, H. Lulka, N. Hanoun, M. Dufresne, A. Meulle, A. Vignolle-Vidoni, L. Ligat, N. Saint-Laurent, F. Pont, S.Dejean, M. Gayral, F. Martins, J. Torrisani, O. Barbey, F. Gross, R. Guimbaud, P. Otal, F. Lopez, G. Tiraby, and P. Cordelier. 2015 Molecular therapy.23:779-789.Matouk, I., E. Raveh, P. Ohana, R.A. Lail, E. Gershtain, M. Gilon, N. De Groot, A. Czerniak, and A. Hochberg. 2013. International journal of molecularsciences. 14:4298-4316.Sidi, A.A., P. Ohana, S. Benjamin, M. Shalev, J.H. Ransom, D. Lamm, A. Hochberg, and I. Leibovitch. 2008. The Journal of urology. 180:2379-2383.

Injection to animal throughdifferent routes of administration

15 min at RT

1. Nucleic acid dilutionin 5 % glucose

2. in vivo-jetPEI® dilution in 5 % glucose

3. Addition ofin vivo-jetPEI® solutionon the nucleic acid solution

Retro-orbital injection (i.v.)

Nasalinstillation

Nebulization

Intraperitoneal injection

Intracerabralinjection

Caudal injection

Intrathecalinjection

Topicalapplication

Intraportalinjection

Intravesicalinjection

Intratumoralinjection

Integrity of nucleic acid over time

D-28

D-21

D-14

D-7

D-3

D-1

Time ofcomplexation

62.8 ± 4.66 nm

36.7 mV

56.4 ± 5.07 nm

32.7 mV

49 ± 7.39 nm

34.2 mV

40.3 ± 7.55 nm

ND

50.8 ± 10.8 nm

30.8 mV

66.3 ± 7.38 nm

34.5 mV

si

Electrophoresis gel without SDS

D-0 D-3 D-7 D-14 D-21 D-28D-1

siRNA/in

vivo-jetPEI®

complexes

Uncomplexed

siRNA

siRNA

released from

complexes

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

1.E+10

RLU

/ m

g o

f p

rote

in

A wide variety of targeted organs depending on the administration route

Complexes were formed using 40 µg or 100 µg of luciferase expressing plasmid and in vivo-jetPEI® at an N/P ratio of 8, in 200 µl or1 ml of 5% glucose and injected either through retro-orbital sinus (IV) or intraperitoneally (IP), respectively. 24 hours after injection,different organs were extracted and luciferase expression was measured or live imaging was performed using IVIS system (PerkinElmer).

I.V. I.P.

Bonnet et al., 2008

in vivo-jetPEI® for clinical trials in Human

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Negative control LPS siRNA in vivo-jetPEI® siRNA+in vivo-jetPEI®

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LTNF-alpha

IL-12/IL-23

IFN-gamma

IL-6

0100020003000400050006000700080009000

LDH ASAT (GOT) ALAT (GPT) ALP

U/I

Negative Control

CCl4

Poly I:C/in vivo-jetPEI®

siRNA

in vivo-jetPEI®

siRNA/in vivo-jetPEI®

Dose response survival study of siRNA/in vivo-jetPEI® complexes

Mice were treated via intravenous injection with increasing amounts of siRNA delivered with in vivo-jetPEI® at an N/P ratio of 12.5,8 and 6 (n=6 per group). Percentage of survival is represented depending on the amount of siRNA.

Bonnet et al., 2013

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Amount of injected siRNA (mg/kg)

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- SDS + SDS Sample 1 2 3 1 2 3

Without serum

With 50% serum (4h)

LD 50

I.V.

I.P.

si D-0 D-3 D-7 D-14 D-21 D-28D-1

Electrophoresis gel with SDS