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`PROTIVA - EXHIBIT 2005
`Moderna Therapeutics, Inc. v. Protiva Biotherapeautics, Inc. IPR2018-00739
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`& © 1997 Nature Publishing Group hitp:/Avww.nature.com/naturebiotechnology
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`trimethylammonium propane (DOTAP)and
`cholesterol that is another step forward in
`improving gene expression in vivo.
`The difficulties involved with the intra-
`venous use of cationic liposome-DNA com-
`plexes arise as a result of their underlying
`transfection mechanism. Cationic liposomes
`form complexes with the negatively charged
`DNAvia charge interactions. Optimal trans-
`fection of the liposome-DNA complex relies
`on the presence of excess positive charge,
`which is required for an efficient interaction
`with the negatively charged cell membrane.
`Neutralization of excess positive charge in
`the liposome-DNA complex by negatively
`charged serum proteinsis likely to result in a
`decreased transfection efficiency. Also, DNA
`might be released from liposome-DNA com-
`plexes by anionic molecules in the serum,
`rendering DNA more susceptible to enzy-
`matic degradation’. Furthermore,
`serum
`proteins can induce an aggregation of lipo-
`some~DNA complexes,
`leading to a rapid
`clearance of liposome-DNA complexes from
`the blood by the reticuloendothelial system
`(RES), These problems, together with others,
`severely limit the intravenous application of
`cationic liposome/ DNA complexes.
`Recently, it has been demonstratedthat the
`serum sensitivity of liposome-DNA complex-
`es can be resolved in several different ways
`(summarized in Table 1). Several important
`conclusions can be drawn from thesestudies.
`First, excess cationic charge in the complex
`with a charge ratio (+/-) ranging from about 2
`to more than 10 seemsto be required foreffi-
`cient gene transfer’’. This may be due to the
`fact that excess amounts of lipids can over-
`come the neutralization effect of the serum
`proteins. Second, a colloidally stable structure
`seems to enhance intravenous gene delivery.
`For example, cholesterol has been shown in
`several studies to enhance gene expression in
`vivo, whereas
` dioleoylphosphatidylethan-
`olamine (DOPE) significantly decreases the
`transduction efficiency of the liposome- DNA
`complex”. This is in contrast to in vitro
`
`transfection, where DOPEis found to improve
`gene
`expression
`in many studies. The
`enhancementoftransfection in vivo by choles-
`terol may be due to its ability to stabilize the
`liposome bilayer. Finally, condensation of
`DNAis also important for a high level of gene
`expression in vivo.
`In the present study, Templetonet al. use
`a liposomal formulation with a uniquestruc-
`ture in which DNAis condensed in the inte-
`rior of invaginated liposomes between two
`lipid bilayers and demonstrate high-level
`expression of the packaged genein vivo. This
`is in agreement with a recent study in which
`
`Templetonet al. use a lipo-
`somal formulation with a
`unique structure in which
`DNAis condensedin the
`interior of invaginated lipo-
`somes between twolipid
`bilayers and demonstrate
`high-level expression of the
`packagedgenein vivo.
`
`the inclusion of protamine sulfate was shown
`to enhance the in vivo activity of the lipo-
`some —DNA complex’. In the latter study,
`plasmid DNA was partially condensed by
`protamine and the protamine~-DNA com-
`plex then interacted with liposomes to form
`small particles with a size about 100-200
`nm’.
`Intravenous administration of liposome
`-DNA complexes can bring about gene
`expression in manytissues, including those
`of the heart, lung, liver, spleen, and Kidney.
`Although the level of gene expression varies
`from study to study,
`the lung invariably
`shows the highest expression levels and
`
`Table 1. Parameters to be considered whendevising a liposome-DNA complexfor
`intravenous administration.
`Parameter
`Importance
`Reference
`Cationic lipid
`Choice oflipid critical: Notall
`lipids equal, some better than others
`Cholesterol more efficient
`than DOPE
`
`Helperlipid
`
`3,4,6,7
`
`Charge
`DNA packaging
`Toxicity
`Stability
`Repeated injection
`Transgene expression
`level
`Duration of expression
`Target
`
`Higher(+/-) ratio enhancesdelivery
`Condensation of DNA enhances delivery
`Only toxic at high doses
`A few weeks to a few months
`Possible after a recovery period
`Picogram to nanogram gene product
`per milligram of tissue
`A few weeks to a few months
`Lung mostefficient target organ
`
`NATURE BIOTECHNOLOGY VOLUME15 JULY 1997
`
`ont
`2,4,5,7
`5-7
`4,5,7
`6
`2-7
`
`2;9,0
`2-f
`
`ANALYSIS
`
`type
`endothelial cells are the major cell
`transfected. This may be due to a “first-pas-
`sage effect” because the lungis thefirst capil-
`lary bed
`the
`liposome-DNA complex
`encounters after intravenous administration.
`Indeed, gene expression in the lung has been
`shown to decrease 100-fold when the lipo-
`some—DNAis injected into the portal vein’.
`Therefore,
`targeted gene delivery to other
`organs via intravenous administration of
`cationic liposome-DNA complexes often
`proves difficult. In the study by Templeton et
`al.,
`incorporation of a targeting ligand,
`asialofetuin, increased gene expression in the
`liver by sevenfold. Nevertheless, the level of
`gene expression in this organ wasstill much
`lower than that in the lung. Thus, the goal of
`tissue-specific
`targeting via
`intravenous
`administration of cationic liposome-DNA
`complexes remains unmet.
`A significant advantage ofliposomal vec-
`tors over viral vectors is their low immuno-
`genicity, which allows repeated injections in
`order to achieve long-term gene expression.
`One surprising observation from a recent
`study is that there is an unresponsive or
`poorly responsive period of about two weeks
`between the two injections during which
`repeated injections are ineffective’. At pre-
`sent, it is not known whether this effect is
`toxicity-related or due to other inactivation
`mechanisms. Onealternative to this problem
`is the use of a novel plasmid DNA with an
`extended half-life, as reported by Thierry et
`al.’ In their study, gene expression lasted for
`about 3 months upona single injection of an
`episomally replicative DNA plasmid com-
`plexed with cationic liposomes.
`Progress in the development ofliposomal
`vectors for intravenous gene delivery repre-
`sents a good beginning, but there is a long
`way to go before it can be translated into
`clinical applications. More toxicity studies
`are required. Also, much remains to be
`learned about the interactions of liposome
`-DNA complexes with serum proteins and
`other components in circulation. On the
`basis of experience of developing “stealth”
`liposomes, the design of an effective and tar-
`get-specific colloidal delivery system for
`DNAwill require detailed study of numerous
`interactions with blood components.
`
`
`
`1. Zhu, N., Liggitt, D., Liu, ¥., and Debs, R. 1993. Science
`261:209-211.
`2. Thierry, A.R. et al. 1995. Proc. Natl, Acad. Sci. USA
`92:9742-9746.
`3. Liu, ¥. et al. 1997. Nature Biotechnology 18:167-173.
`4. Hong, K.L., Zheng, W.W.,
`Baker, A,
`and
`Papahadjopoulos, D. 1997. FEBS Lett. 400:233-237.
`§. Ui, S. and Huang, L. 1997. Gene Ther. In press.
`6. Liu, F, Qi, H., Huang, L., and Liu, D. 1997. Gene Ther.
`In press.
`7. Templeton, N.S., Lasic, D.D., Frederik, RM., Strey,
`H.H., Roberts, D.D., and Paviakis, G.N. 1997. Nature
`Biotechnology 15:647-652.
`8. Xu,
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`and Szoka,
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