JC Virus-like particles

An effective and specific delivery system

with simplified retargeting

J. Gruber – Biovaria 2017

The Drug Delivery Problem

New Options, e.g. RNAi or CRISPR

Need: Efficient, safe and targeted delivery

• Drugs

• RNA

• DNA

2

Directed delivery (cell type / tissue)

Use in vivo, ex vivo, in vitro:

Specificity

Toxicity

Immunogenicity

Efficiency

So far: no suitable delivery-technology

available

Carrier requirements (nanoparticles)

size shape charge

Blanco et al., Nat Biotech, 2015

Our Solution: Targeted Virus-Like Particles (VLPs)

VLPs – the basics of a platform technology

Capsid Protein VP1 of human Polyoma JC Virus

spontaneous assembly and controlled disassembly of VLPs

perfect circulation size (appr. 40 nm diameter)

multitude of target cells (uptake via 5HT2 receptors and NeuNac LSTc)

crosses the blood-brain-barrier for CNS delivery

nuclear delivery of DNA in resting cells

3

JCV VP1 VLP

• Delivers cargo into many

cell types.

• Enhanced NLS

increases nuclear

transport.

VLP:

Production/

purification/

cargo loading

Retargeting of VLPs

4

e.g. single chain antibody

(scFv) against Her2/neu

VLP-attachment(covalent via crosslinker)

glycosylated TM

(direct or indirect)

VLP-attachment(spontaneous via affinity)

• Retargeted VLP

• Delivers cargo into selected cell types,

e.g. Her2/neu positive cancer cells

• Native tropism of JCV is blocked by TM

Attachment of Targeting molecule (TM)

(+) covalent TM

(+) well established

(-) multiple steps

(-) free components

(+) affinity driven

(+) one step

(+) free components

(-) TM manipulation

Method 1:

covalent via VLP-exposed lysine residue

Method 2:

by strong affinity to LSTc-glycosylated TM

Applications – Research Tool in vitro & ex vivo

5

RNA Delivery DNA Delivery

HEK293 cells

(Recipient cell)Donor cell (Raji)

siRNA

(direcetd) delivery of synthetic siRNA

Broad variety of cell types

Primary cells (from brain to bone)

Exosome loading

exosomes

cortical neurons osteoblasts

Gene expression from linear DNA-

constructs

Efficient gene transfer

Genome editing

more on poster

(CRISPR/Cas9) GFP-gRNA

Applications – Preclinics in vivo

6

Systemic siRNA Delivery

• Systemic siRNA-delivery in rodents

• Efficient gene silencing in long bones

• Repeated injections (i.p.)

• Apathogenic

• Effective dose 0.5 mg/kg bodyweight

Immunogenicity

• Six immune-competent Rhesus macaques

• Four repeated injections (i.v. or i.m.)

• Temporary increase of early activation

marker (CD69+ lymphocytes)

• Low IFN- levels

• Transient IgM response, no IgG

• Stimulation Index – no cellular response

Commercial Potential

Targeted Drug- or Gene-Delivery for clinical applications

- Indications: Osteoporosis and fracture healing, cancer, infectious diseases

(HIV-1)

Target identification & validation (e.g. RNA Interference, CRISPR/Cas9)

- platform technology for multiple purposes in research, biotech and pharma

Transduction/Transfection in vitro, ex vivo, in vivo

- cultured cells, tissue explants, organoids, animal studies

- TMs for T-cells, B-cells, MSCs, hepatocytes, neurons, osteoblasts, various

cancers (colorectal, breast, lung, glioblastoma), lung slices, brain slices

Models

- CNS/neurodegenration, neuro-optogenetics, infectious diseases, cancer and

metabolic diseases

7

Clin

ics

R&

D T

oo

l

Development status

8

The technology is ready for in vitro and in vivo delivery of nucleic

acids (e.g. siRNA, miRNA or DNA expression cassettes)

A proof of concept study for the VLP technology was performed with

siRNA delivery in a rodent model (part 1 published, part 2 with 10

repeated injections in ovariectomized rats as osteoporosis model

succeeded)

A limited repertoire of targeting molecules including scFv for

Her2/neu is available for testing

Production and purification protocols for VP1 and targeting

molecules set for lab-scale and animal testing

Alternative Delivery Technologies

pro:

well established, ease ofproduction, efficient, targeting

contra:

safety, immunity, requiresexpression

Viruses

(Lentivirus, Adeno- or

Adenoassociated)

pro:

easy and cheap, many materials(dendrimers, carbon, metalcomplexes etc.)

contra:

(cyto-) toxicity, biocompatibility, limited cargoclasses

Nano-particles

pro:

in vivo tolerance, high loadingcapacity, limited targeting

contra:

loading efficiency, circulation

Lipo-plexes,

vesicles, Exosomes

9

VLPs

• no toxicity

• broad cargo repertoire

• efficient retargeting

• easy and efficient

loading

• versatility of cargo and

targeting

• low dosage required

• no viral genome

components

• not immunogenic

• safe in vivo use

IP status and Freedom-to-Operate

Patents protecting the nucleic-acid loaded JCV-VLP are granted in

US and JP, and pending in EP and CA.

Patent application for sequence-optimized VLPs with enhanced

transduction efficiencies in non-dividing cells is pending.

Patent application protecting the affinity-attached retargeting

molecules and uses has recently been filed.

The German Primate Center owns IP for VLP utilizations, different

cargos or targeting molecules may affect the FTO

10

Risks, Bottlenecks, Resources

Risks

Immune responses of TM

Stability of targeted complexes

in vivo

Dissemination failure

11

Bottlenecks

Production capacities

Systematic analysis of a broad

range of TMs (e.g. scFv library)

Manpower

Resources needed

Increased production capacities & GMP

Additional staff (technical)

One year, 500k Eur (VLP production & TM screen for 15 targets)

Acknowledgements

12

Young Leaders in

Science

DPZ

Astrid Backhaus

Ellen Eckermann-Felkl

Angelina Schuder

Lara T. Schiller

Stefan Schneider

Rafael Rinaldi Ferreira

Nicolas Lemus

Kai Böker

Manfred Eberle

LMU (Munich)

Lars König

Lorenz Kocheise

IDCBIS (Bogota)

Gustavo Salguero Lopez

Monica Liliana Cruz Barrera

UMG

Trauma-Surgery

Stephan Sehmisch

Marina Komrakova

Daniel B. Hoffmann

Arndt F. Schilling

Visceral Surgery

Jochen Gaedcke

Azadeh Azizian

Jessica Eggert

Evotec

Arnd Steuernagel

Alina Mosblech

European Neuroscience Institute (ENI)

Camin Dean

Markus Stahl

Charles Gilbride