inhibition of hepatitis b virus replication using synthetic antiviral rna interference activators

256 Pages·2016·13.46 MB·English

INHIBITION OF HEPATITIS B VIRUS
REPLICATION USING SYNTHETIC
ANTIVIRAL RNA INTERFERENCE
ACTIVATORS

Musa Donald Marimani

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand in
fulfilment of the requirements for the degree of Doctor of Philosophy.

Johannesburg 2016
Declaration

DECLARATION
I, Musa Donald Marimani declare that this thesis is my own work. It is being submitted for
the degree of Doctor of Philosophy in the University of the Witwatersrand, Johannesburg.
It was not submitted before for any degree or examination at this or any other University.

..............................................................
25 May 2016

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Dedication
DEDICATION
This work is dedicated to members of the Marimani family (Khoza clan) of the Shangaan
nation and my friends:
UMagudu lowo kade bamtsho abeNguni bathi:
uMagudu uyakhanya,
ngoba waqinisa inkanyezi ya khanyisa intaba kaMagudu.
Makhonkotha ebusuku, kuvuka abathakathi,
ngoba engu Mabona.
Mabona kude owabona impi igqamuka, ngaphesheya komfula eNkomazi.
Umude kodwa kawumdaka, unjenge zinhlanga zamabele,
uyakhalipha, ngoba usika hlangothi zombili,
njengo mkhonto waseChaimite.
Mlotshwa, Mkhathini, Mlilo, Mabona, Nomageja,
Sokhabase, Skotana, Ntshamase, Ndlandla!

Ripanga raku xeka homu rixeka MaKhosa.
Magudu, Phukwani, tinhlanga ta mavele enkanyini.
Hi vaka Marimani, hi vaka Magigwana, hi vaka Magudu, hi vaka Mavona, hi vaka Khosa.
Hi vaka ntsanda ava tluli, ku tlula Khosa a vonaka. Anga Mavona na Magigwana.
Vaku: Mangatlu ava hungeli, va hungela tuva xingana vutoya. Hi swigwigwigwigwi vafa
vatele! I Maputukezi, vaku: Obrigado Magigwana, aluta continua, Saudação lenda
Shangaan! I nghundu ya Magigwana ya ku phazamisa Maputukezi ka Gaza. Wena waka
mkhonto awu goveki, ku goveka xibamu xa Maputukezi. EBileni ka Masiya byi virile. Ka
Magudu eXinavana enkanyini, incambala ya Mavona eKhoseni! Hi vahloti vaku dlaya
tinghala hi thlarhi. UMabona omuhle, namehlo akhe. Lawo khanya nok` bona
ebum`nyameni.
Marimani! Khoza!
Mabona! Khoza!
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List of publications and presentations
LIST OF PUBLICATIONS AND PRESENTATIONS

Publications:
1. Jolanta Brzezinska, Jennifer D’Onofrio, Maximilian C.R. Buff, Justin Hean, Abdullah
Ely, Musa Marimani, Patrick Arbuthnot, Joachim W. Engels (2012). Synthesis of 2’-
O-guanidinopropyl-modified nucleoside phosphoramidites and their
incorporation into siRNAs targeting hepatitis B virus. Bioorganic and Medicinal
Chemistry 20: 1594–1606.

2. Musa D. Marimani, Abdullah Ely, Maximilian C.R. Buff, Stefan Bernhardt, Joachim
W. Engels, Patrick Arbuthnot (2013). Inhibition of hepatitis B virus replication in
cultured cells and in vivo using 2’-O-guanidinopropyl modified siRNAs.
Bioorganic and Medicinal Chemistry 21: 6145-6155.

3. Musa Marimani, Justin Hean, Kristie Bloom, Abdullah Ely and Patrick Arbuthnot
(2013). Recent advances in developing nucleic acid-based HBV therapy. Future
Microbiology 8 (11): 1489-1504 (Invited Review).

4. Maximillian C.R Buff, Stefan Bernhardt, Musa D. Marimani, Abdullah Ely, Joachim
W. Engels, Patrick Arbuthnot (2015). Use of Guanidinopropyl-Modified siRNAs to
Silence Gene Expression. Methods in Molecular Biology 1218: 217-249 (Invited
paper).
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List of publications and presentations
5. Musa D. Marimani, Abdullah Ely, Maximillian C.R Buff, Stefan Bernhardt, Joachim
W. Engels, Daniel Scherman, Virginie Escriou, Patrick Arbuthnot (2015). Inhibition
of replication of hepatitis B virus in transgenic mice following administration of
hepatotropic lipoplexes containing guanidinopropyl-modified siRNAs. Journal of
Controlled Release 209: 198-206.

Presentations:

1. University of the Witwatersrand Biennial Health Sciences Research day and
Postgraduate Expo, 17 September 2014, Wits University, Johannesburg, South
Africa. Poster: Musa D. Marimani, Abdullah Ely, Maximilian CR. Buff, Stefan
Bernhardt, Justin Hean, Joachim Engels and Patrick Arbuthnot. Silencing of hepatitis
B virus replication using guanidinopropyl modified siRNAs (MCB-P-18)

2. European Society of Gene and Cell Therapy (ESGCT), 25-28 October 2013,
Palacio Municipal de Congresos, Madrid, Spain. Poster: Musa D. Marimani, Abdullah
Ely, Maximilian CR. Buff, Stefan Bernhardt, Justin Hean, Joachim Engels and Patrick
Arbuthnot. Inhibition of hepatitis B virus replication using guanidinopropyl modified
siRNAs (P004)

3. Department of Molecular Medicine and Haematology Presentation, 23 October
2013, Departmental Audio-visual Seminar Room, Wits University, Johannesburg,
South Africa. Oral: Musa D. Marimani, Abdullah Ely, Maximilian CR. Buff, Stefan
Bernhardt, Justin Hean, Joachim Engels and Patrick Arbuthnot. Inhibition of hepatitis
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List of publications and presentations
B virus replication in cultured cells and in vivo using 2’-O-guanidinopropyl modified
siRNAs

4. Wits 5th Cross-Faculty Postgraduate Symposium, 01 August 2013, Wits University,
Johannesburg, South Africa. Oral: Musa D. Marimani, Abdullah Ely, Maximilian CR.
Buff, Stefan Bernhardt, Justin Hean, Joachim Engels and Patrick Arbuthnot. Inhibition
of hepatitis B virus replication in cultured cells and in vivo using 2’-O-
guanidinopropyl modified siRNAs

5. The Molecular Biosciences Research Thrust (MBRT) Research Day, 05 December
2012, Wits University, Johannesburg, South Africa. Poster: Musa D. Marimani,
Abdullah Ely, Maximilian CR. Buff, Stefan Bernhardt, Justin Hean, Joachim Engels
and Patrick Arbuthnot. Silencing of hepatitis B virus replication in vitro using novel 2’-
O-guanidinopropyl modified siRNAs

6. Wits 4th Cross-Faculty Postgraduate Symposium, 19 and 22 October 2012, Wits
University, Johannesburg, South Africa. Poster: Musa D. Marimani, Abdullah Ely,
Maximilian CR. Buff, Stefan Bernhardt, Justin Hean, Joachim Engels and Patrick
Arbuthnot. The use of 2’-O-guanidinopropyl modified siRNAs to silence hepatitis B
virus replication in cultured cells

7. International Meeting on Molecular Biology of Hepatitis B Viruses, 22-25
September 2012, Oxford University, Oxford, England. Poster: Musa D. Marimani,
Abdullah Ely, Maximilian CR. Buff, Stefan Bernhardt, Justin Hean, Joachim Engels
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List of publications and presentations
and Patrick Arbuthnot. Inhibition of hepatitis B virus replication using
guanidinopropyl modified siRNAs (P-170)

8. University of the Witwatersrand Biennial Health Sciences Research day and
Postgraduate Expo, 19 September 2012, Wits University, Johannesburg, South
Africa. Poster: Musa D. Marimani, Abdullah Ely, Maximilian CR. Buff, Stefan
Bernhardt, Justin Hean, Joachim Engels and Patrick Arbuthnot. Inhibition of hepatitis
B virus replication using guanidinopropyl modified siRNAs

9. South African Society of Biochemistry and Molecular Biology
(SASBMB)/Federation of African Societies of Biochemistry and Molecular
Biology (FASBMB), 29 January-01 February 2012, Champagne Sports Resort
Drakensberg, South Africa. Poster: Musa D. Marimani, Abdullah Ely, Maximilian CR.
Buff, Stefan Bernhardt, Justin Hean, Joachim Engels and Patrick Arbuthnot. Inhibition
of hepatitis B virus replication using synthetic anti-viral RNA interference activators

10. Molecular Biosciences Research Thrust (MBRT) Research Day, 07 December
2011, Wits University, Johannesburg, South Africa. Poster: Musa D. Marimani,
Abdullah Ely, Maximilian CR. Buff, Stefan Bernhardt, Justin Hean, Joachim Engels
and Patrick Arbuthnot. Silencing of hepatitis B virus replication using 2’-O-
guanidinopropyl modified siRNAs

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Abstract
ABSTRACT
Chronic liver infection by hepatitis B virus (HBV) may lead to devastating clinical
conditions that include hepatocellular carcinoma and cirrhosis. Approved antiHBV drugs
do not completely eradicate the infection, leading to continued viral persistence in infected
individuals. Inhibition of HBV replication using synthetic activators of RNA interference
(RNAi) may provide a feasible strategy of developing superior antiviral drugs. The aim of
this study was to evaluate the therapeutic utility of novel 2’-O-guanidinopropyl (GP)
modified synthetic small interfering RNAs (siRNAs) to counter HBV replication in
cultured mammalian cells and mice. Initially, single GP moieties were placed at different
nucleotide positions of the guide strand of a potent antiHBV siRNA. Some GP-modified
siRNAs enhanced antiHBV activity in vitro following transient transfection of Human
hepatoma 7 (Huh7) cells with siRNAs and pCH-9/3091, a replication competent HBV
target plasmid. These siRNAs inhibited the secretion of Hepatitis B surface antigen
(HBsAg) by up to 95% in Huh7 cells. The level of knockdown exhibited by some modified
siRNAs was statistically significant relative to that displayed by unmodified siRNA3
which achieved HBsAg silencing of 73%. Additionally, modified siRNAs were also
capable of reducing RNA containing the X sequence in vitro by 88-93%. Impressively,
some of these knockdown levels were statistically significant when compared to
unmodified siRNA3, which achieved HBx knockdown of 83%. Quantitation of interferon
(IFN) response genes by reverse transcription quantitative polymerase chain reaction (RT-
qPCR) and evaluation of cell viability by 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay revealed no evidence of innate immune
stimulation or cytotoxicity in cultured cells, respectively.
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Abstract
Modified siRNAs also displayed moderate stability in 80% foetal calf serum (FCS). Target
specificity was markedly improved by GP-modified siRNAs, especially those with seed
modifications (comprising nucleotide position 2 to 8 from the 5’ end of the guide strand).
The siRNA-mediated mRNA cleavage product was detected from transfected cells using 5’
Rapid Amplification of cDNA ends (5’ RACE).

In the hydrodynamic mouse model, co-injection of GP-modified siRNAs and HBV
plasmid vector led to HBsAg suppression of approximately 80-92% at day 3 and 77-96%
at day 5 post-administration. The HBV knockdown levels observed at day 3 were
statistically significant when compared to those displayed by unmodified siRNA3 which
achieved HBsAg silencing of 58% during the same time frame. Furthermore, both sets of
siRNAs also suppressed the number of circulating viral particle equivalents (VPEs) by 88-
90% at day 3 post-injection. HBV silencing efficacy of 70-75% and 65% was achieved by
modified and unmodified siRNAs, respectively at day 5 post-administration.

Finally, antiHBV efficacy of GP-modified siRNAs was tested in HBV transgenic mice
following delivery of these RNAi effectors using cationic polyglutamate (PG) adjuvant
liposomes. Both groups of antiHBV siRNAs effected HBsAg knockdown that ranged from
70-86% at day 3 to 7 post-administration as siRNA lipoplexes in HBV transgenic mice. In
contrast to the unmodified siRNA3, GP-containing siRNAs achieved durable HBsAg
silencing of 70% at day 14 post-administration, while the unmodified siRNA3 displayed a
shorter duration of activity. As with HBsAg data, the GP-modified siRNAs also displayed
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Abstract
silencing efficacy that was similar to the unmodified siRNA, reducing the number of
circulating VPEs by 95% from day 3 to 7 post-injection. However, the unmodified siRNA3
lost efficacy by day 14 post-administration, while the GP-modified siRNAs displayed
prolonged suppression by reducing the number of circulating VPEs by 75% during the
same time interval. Intrahepatic RNA levels were also assessed in transgenic mice, in
which GP3 siRNA3 significantly suppressed surface and core RNA levels by 40 and 42%,
respectively at day 18 post-injection. The unmodified siRNA3 suppressed surface RNA
levels by 20% and core RNA levels by 25% at day 21 post-administration. Furthermore,
GP4 siRNA3 silenced both surface and core RNA levels by 42% during the same time
period. Additionally, intrahepatic RNA quantitation revealed no induction of IFN response
genes by either unmodified or GP-modified siRNAs. In contrast to mice that had received
GP-modified siRNAs, significant induction of proinflammatory cytokine release was
observed in mice treated with unmodified siRNAs. The siRNA-mediated mRNA cleavage
product was also detected from liver samples following 5’ RACE analysis. Neither GP-
modified nor unmodified siRNAs significantly induced toxicity in injected mice.
Collectively, our data provide evidence that utilisation of GP-modified siRNAs and an
efficient hepatotropic non-viral delivery system may be used as a strategy to counter
chronic HBV infection.

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