Cell-permeable miniature proteins: better delivery … · Cell-permeable miniature proteins: better delivery for better medicine “Imagine a world in which protein therapeutics could

Cell-permeable miniature proteins: better delivery for better medicine

“Imagine a world in which protein therapeutics could be delivered efficiently to the cytosol and nucleus.”

Alanna SchepartzSterling Professor

Department of Chemistry

A novel platform technology applied (first) to an illness with no disease-modifying therapy: Type 1 Citrullinemia (CTLN I)

Rebecca WissnerPostdoctoral Associate

Department of Chemistry

We discovered cell-permeable miniature proteins

• CPMPs reach cytosol and nucleus with efficiencies as high as 75%

• Efficiency higher than all other ‘CPPs’ tested

• Multiple cell types• Deliver active payloads 10 – 32 kD

• High and tunable serum stability

• Non-toxic; genetically encodable; easily manufactured

• Key difference: CPMPs utilize well-

defined, non-destructive mechanism • Fundamental patents and applications covering both scaffold and delivery

• Potential partners already engaged

quantifies endosomal escape

Using a CPMP to deliver AS and correct ‘inborn error of metabolism’

Type 1 citrullenemia is an incurable disease

Results from deficiency or absence of the urea cycle enzyme argininosuccinatesynthetase (AS)

severe AS deficiency results in hyperammonemia and irreversible neurological damage, coma, or death

The Problem 1:57,000 affected worldwide;

19,000 patients in US/Europe no disease modifying therapy mutations suggests activity

>10% would be disease-modifying

@ $350K/patient/year, 15% treated = $1B/year.

Blavatnik: success validates platform for therapeutic indications where delivery to cytosol is critical

The Market

Favorable competitive landscape

Cure disease

Deliver enzyme

Broad scope

High stability

Non-toxic

High deliveryefficienc

y

CTLN

1 tr

eatm

ents

Buphenyl® or Ravicti® X X X – – –

Shire, PhaseRx ✔ X X X – X

SynLogic ✔ ✔ X – – –

plat

form

s Aileron, Bicycle X X X ✔ ✔ ✔TrojanTech, Xigen, Portage, Cellivery

✔ ✔ ✔ X X X

Cell-permeable miniature proteins

✔ ✔ ✔ ✔ ✔ ✔

Timeline, Funding and Milestones• Express CPMP-AS fusions/controls• Evaluate uptake and trafficking efficiency in

SK-HEP-1 cells• Monitor reversal of AS deficiency• Milestone: Successful

overexpression/purification; enhanced cytosolic trafficking; AS deficiency reversal

• Back-up: Different disease-relevant urea cycle enzyme or PKU Q3-4 2017

• Establish in vitro PK and metabolism of CPMP-AS fusions/controls

• Evaluate plasma stability, protein binding• Compare results to those of FDA-approved

biologics• Milestone: acceptable stability (t1/2 > 30

min); PPB comparable to Fabrazyme®; establish fundamental PK

• Back-up: Judicious changes in CPMP sequence and/or linkage to AS Q1 2018

• Evaluate top CPMP-AS fusion in foldmouse model of human CTLN1 (iv)

• Assess lifespan, weight, length, and coat density, and plasma ammonia and citrulline.

• Liver and peripheral white blood cell samples will be collected and stored Q1-2 2019

• Milestone: Inflection point–Demonstration that CPMPs can deliver an active enzyme to the cytosol of an animal to reverse the effects of a serious metabolic disease

Objective 1

Objective 2

• Establish in vivo PK and biodistributionCPMP-AS fusions/controls C57BL/6 mice

• Milestone: presence in plasma; acceptable distribution to liver

• Back-up: Judicious changes in CPMP sequence and/or linkage to AS Q3 2018

Objective 3

Objective 4

Cell-permeable miniature proteins: delivering better medicine

More about the competition

Current treatment: Buphenyl® or Ravicti® delivered orally 3x daily; Combined sales $175 M

Three products in development Shire: mRNA replacement therapy. Detect ASS 1 week PD (1 mg/kg IV) in

WT mice; plasma NH3 lowered for 1 day PD in fold mouse. ”urea cycle defects allow rapid clinical proof-of-concept and compelling market opportunity”; delivery via lipid nanoparticles

PhaseRx: mRNA replacement therapy. PRX-ASL listed as pre-clinical. delivery via lipid nanoparticles. Claim to have corrected Ornithine Transcarbamylase deficiency in mice reported in May 2017. deliver mRNA into inert lipid nanoparticle – no fusogenic lipid. ILP simply gets the mRNA to the liver. Licensed from UWash. Covered with GalNAc.

Synlogic-microbiome strategy. Have patent on live engineered bacterium modified to assimilate ammonia for the potential treatment of hyperammonemia-based diseases such as urea cycle disorders (UCDs) and hepatic encephalopathy.

Current/pre-clinical CTLN1 treatments

More about the competitionCompetitive CPP technologies

Medtrack identified 9 products under development; most (7 of 9) for cancer.

No evidence that any CPP company is focusing publicly on UC disorders Xigen: private, Swiss, “peptides for use against inflammation”; D or D/L-

peptides. XG-102/104 (JNK inhibitor) Portage: public, Canadian, humanized version of antennapaedia peptide

called CellPorter®; PPL-003 developed for Dry Eye Disease Cellivery: hydrophobic signal sequence derived from membrane-

translocating sequence in fibroiblast growth factor 4 Aileron: AKRN-6924 is a stapled peptide that inhibit p53•hDM2 and

p53•hDMX interactions. Phase I results expected June 2017 (ASCO) Bicycle Therapeutics: Large, cyclized peptides generated by phage

display; not clear they are actually cell-penetrant CPMPs have higher stability and efficiency than Xigen, Portage, or

Cellivery. CPMPs have broader scope than Aileron or Bicycle Therapeutics

Timeline, Funding and Milestones

Urea cycle and its role in diseasemitochondria

cytosol

Key data: CPMP delivery of AS27 surpasses Tat

AS27, a 10 kD monobody targeting the Bcr-Abl SH2

domain, reaches the cytosol only when delivered by ZF5.3 CPMP

CPMP Cytosolic delivery of AS27 is at

least twice as efficient as that provided by Tat

Evidence that CPMPs can deliver 20-35 kD enzymes to the cytosol

Delivery of enzyme #1 (20 kD)

Delivery of enzyme #2 (35 kD)

Cell-permeable miniature proteins possess many desirable properties

Most ‘CPPs’•toxic•unstable•retained in

endosomes•poorly defined

mechanism•little reaches

cytosol intact

CPMPs•not toxic (Smith et al. 2008)•high and tunable stability

(Appelbaum et al. 2012)•efficient endosome release

(LaRochelle et al. 2015)•well-defined mechanism-

non-destructive movement •more reaches cytosol intact

and active