A. UNTREATED B. GFP-AAV

C. DRS1-AAV

D. DRS2-AAV

Finding 3: Mouse liver demonstrated Casp-9 overexpression in HBV-infected hepatocytestreated with Hijack RNA AAVØ Casp-9 IHC staining showed extensive casp-9 overexpression in DRS1-AAV and DRS2-AAV treated, but not in GFP-

AAV treated or untreated mice liver tissue.

Hijacking HBV Pol to Selectively Induce Apoptosis in Infected Hepatocytes In Vivo:A Novel Approach for Potential Treatment or Cure

Serhat Gümrükcü1 , Tung X Nguyen1,2, Michael Bobardt3, Joseph Kuo3, Phillip Musikanth1, Philippe Gallay3

1Seraph Research Institute, Los Angeles, CA; 2Enochian Biosciences, Los Angeles, CA: Department of Immunology & Microbiology, 3The Scripps Research Institute, La Jolla, CA

Ø Despite an effective vaccine, approximately 5 percent of the world’s population chronically carryhepatitis B virus (HBV) and nearly 1 million people die each year.

Ø Several promising avenues for treatment have been pursued including depleting the pool ofcovalently closed circular (ccc)DNA and inhibiting HBV polymerase (HBV pol), which is essentialboth for its reverse transcription and packaging functions.

Ø In this study, rather than trying to inhibit the HBV life cycle, we utilize the virus and cellularmachinery to kill the infected cells as a potential cure.

Ø HBV-transgenic mice, which constitutively express HBV from kidney and liver cells, receivedintraperitoneal injections with 1011 AAV8 particles packaged with Hijack RNA under strong EF1apromoter (DRS1-AAV8) or liver-specific TBG promoter (DRS2-AAV8), or GFP expressingmock/control vector (Fig. 5). Day 14 post-injection, liver and kidney tissues were evaluated.Weekly liver enzymatic activities (ALT and AST) were monitored.

Ø AAV particles were packaged with a novel and proprietary vector construct that expresses a non-functional, non-coding (nc)RNA, HBV “Hijack RNA”, under a strong promoter (Fig. 1).

Ø Open reading frame (ORF) of HBV Hijack RNA is derived from the negative strand of humancaspase-9 (casp-9) coding region, and it is flanked between the HBV epsilon signals (Fig. 2).

Ø HBV epsilon signal is the recognition sequence that is specific to HBV pol. The reversetranscriptase domain of HBV pol (HBV pol/RT) recognizes the Hijack RNA through these sequencesand reversely transcribes it into a double stranded (ds)DNA that codes for casp-9, driven by astrong promoter (Fig. 3).

Ø The dsDNA engages with host polymerases to overexpress casp-9 and induce apoptosis of theinfected cell (Fig. 4A).

Ø In an uninfected cell, without the presence of HBV pol, Hijack RNA would be non-functional andbe degraded (Fig. 4B).

RESULTS

Ø Hijacking HBV pol to express casp-9 induces significant cell death in HBV-infected but not in HBV-uninfected cells in vitro.

Ø Reverse transcriptase and caspase inhibition abrogated cell death, validating the hypothesizedmechanism of action.

Ø Data from in vivo studies demonstrate that HBV Hijack RNA induces apoptosis of HBV-infectedhepatocytes resulting in inflammation and increased liver enzymes.

Ø However, transgenic mouse models are inherently limited to study the potential for HBV curesince it is impossible to eradicate HBV in animals that constitutively expresses the HBV genomein every hepatocyte.

FIGURE 1: THE DESIGN OF THE HBV “Hijack RNA” AAV. Through AAV delivery, the designed non-coding Hijack RNA isexpressed in the target cells.

FIGURE 2: THE STRUCTURE OF THE “Hijack RNA”. Reverse complementary strand of EFS promoter driving humancasp-9 gene flanked between HBV epsilon signal sequences to be reverse transcribed by HBV Pol/RT .

FIGURE 3: HBV “Hijack RNA” reverse transcribed by HBV Pol/RT into dsDNA overexpressing human casp-9.

FIGURE 4: MECHANISM OF ACTION – HYPOTHESIZED MODEL. A: Delivery of the “Hijack RNA” by AAV to HBV-infected hepatocyte induces apoptosis through overexpression of casp-9 utilizing HBV pol, which engages andreversely transcribes the designed Hijack RNA; B: In the absence of HBV pol in the uninfected hepatocytes, theoverexpressed Hijack RNA will undergo degradation, and C: Treatment with HBV pol /RT inhibitors or caspaseinhibitors blocks either reverse transcription of the Hijack RNA or activation of casp-9, both of which are needed totrigger the infected cell death through apoptosis.

BACKGROUND

MECHANISM OF ACTION

METHODS (CONT)

DISCUSSION

Finding 5: Increased liver enzymes in mice treated with HBV Hijack RNA AAV indicatehepatocellular damageØ AST levels increased 2.2 fold and 1.8 fold in mice treated with AAV-DRS1 and AAV-DRS2, respectively compared to

GFP-AAV treated and untreated mice at week 4.Ø ALT levels increased 1.4 fold in both treatment groups compared to GFP-AAV treated and untreated mice.

GFPDistribution - Expression

APOPTOSISEfficacy

APOPTOSISSafety

1 32

GFP rcCASP9 TBG-rcCASP9

5A 5B 5C

FIGURE 5: ENGINEERED CONSTRUCTS AND STUDY DESIGN FOR IN VIVO HBV-TRANSGENIC MOUSE STUDY. Mice were injected intra-peritoneal with 1011 AAV8 particles packaged with test or mock/control vector.

Finding 1: Test vector selectively increased casp-9 levels in HBV infected cellsØ There was a 254% increase in casp-9 levels in the treated HBV-infected, but not uninfected, cells (Fig. 6A).Ø Casp-9 inhibitor salvaged the Hijack RNA-mediated infected cell death as demonstrated by expansion of viable cells

by 10-20% (Fig. 6B).

HepG2

GFP-AAV2-T

reated

HepG2

DRS1-AAV2-T

reated

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reated

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reated

HepAD38

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OPENED : CASP3/7

FILLED: CASP 9

OPENED: CASP 3/7

FILLED: CASP 9

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-48hr

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-144h

r0

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FIGURE 6: Test vector selectively increased casp-9 levels in HBV infected cells

: CASP 3/7

0 1 2 3 40

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DAYS

% C

ELL

DEAT

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HepG2+AAV2-GFP

HepG2+AAV2-TEST

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Huh+AAV2-GFP

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HepG2 H1.3+AAV2-GFP

HepG2 H1.3+AAV2-TEST

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HepAD38+AAV2-TEST

HepG2 H1.3+AAV2-TEST+TDF

HepG2 H1.3+AAV2-TEST+ETV

HepG2 H1.3+AAV2-TEST+Z-VAD-FMK

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Untransfected HepG2+AAV2-GFP

Untransfected HepG2+AAV2-TEST

HBV pol-RT transfected HepG2+AAV2-GFP

HBV pol-RT transfected HepG2+AAV2-TEST

Untransfected Huh+AAV2-GFP

Untransfected Huh+AAV2-TEST

HBV pol-RT transfected Huh7+AAV2-GFP

HBV pol-RT transfected Huh7+AAV2-TEST

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Uninfected NTCP+HepG2+AAV2-GFP

Uninfected NTCP+HepG2+AAV2-TEST

HBV-infected NTCP+HepG2+AAV2-GFP

HBV-infected NTCP+HepG2+AAV2-TEST

Uninfected PHH+AAV2-GFP

Uninfected PHH+AAV2-TEST

HBV-infected PHH+AAV2-GFP

HBV-infected PHH+AAV2-TEST

7A 7C

7B 7D

FIGURE 7: Hijack RNA induced cell death in vitro

Finding 2: HBV Hijack RNA selectively kills HBV-infected or HBV pol expressing cellsØ Mean cell death in a variety of HBV-producing cells was 92% (ranging 88.6% to 95.8%), by day 4 (Fig. 7A-7C).Ø No significant cell death was observed in uninfected cells (Fig. 7A-7C).Ø RT and pan-caspase inhibitors individually prevented cell death in AAV-treated infected cells (Fig. 7D).

METHODSØ Expression of casp-9 were examined in AAV2-treated HepG2 and the HBV-infected HepAD39 cell

lines.Ø HBV-infected and uninfected hepatoma cell lines and primary human hepatocytes (PHH) were

treated with AAV2 particles expressing Hijack RNA “test AAV” or green fluorescent protein (GFP)Ø To validate the mechanism of action, HBV-infected HepAD38 cells were treated with the test AAV

in the presence or absence of RT inhibitors tenofovir or entecavir, casp-9-specific inhibitor Z-LEHD-FMK or pan-caspase inhibitor Z-VAD-FMK (Fig. 4C). Cell viability and proliferation were evaluatedby Chemometec NC-200 and FACS analysis using PI/Annexin V and TUNEL assays daily.

RESULTS (CONT)

A. DRS1-AAV B. DRS2-AAV C. UNTREATED D. GFP-AAV

FIGURE 9: Deep purple staining demarcates neutrophil infiltration in untreated (9A), GFP-AAV control vector-treated (9B) and Hijack RNA AAV vector-treated (9C, 9D) mice.

FIGURE 8: Casp-9 IHC staining of mouse liver tissue at week 2 post-injection of AAV

FIGURE 8: CYTOLOGY ANALYSIS revealed high level of casp-9 (IHC brown stain) in HBV-infected hepatocytes from mice treated with Hijack RNA AAV (8A and 8B) but not in untreated (8C) or GFP-AAV control vector treated mice (8D)

Finding 4: Hijack RNA AAV induced extensive neutrophil infiltration indicating HBV-infectedhepatocyte damageØ Diffuse neutrophil infiltration indicating an inflammatory reaction to HBV-infected hepatocytes in mice treated

with Hijack RNA AAV.Ø Untreated mice or mice treated with GFP AAV control vector exhibited no evidence of inflammation

FIGURE 9: Hematoxylin and Eosin staining of mouse liver sections

FIGURE 10: INCREASE OF LIVERENZYMATIC ACTIVITY 4-WEEK POSTTREATMENT WITH HIJACK RNACARRYING VECTOR AAV

CONCLUSIONS

Ø These results demonstrate a novel mechanism of action for a potential cure for HBV infection.Ø Additional in vitro and in vivo studies are in progress.

IU/L