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Lentiviral*Vector*Purification* Using*Genetically*Encoded*Biotin* Mimic*In*Packaging*Cell*

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Lentiviral*Vector*Purification* Using*Genetically*Encoded*Biotin* Mimic*In*Packaging*Cell* ! ! Lentiviral*vector*purification* using*genetically*encoded*biotin* mimic*in*packaging*cell* ! ! UCL!Cancer!Institute! University!College!London! A!thesis!Presented!for!the!Degree!of!Doctorate!in!Philosophy!! ! ! Leila*Mekkaoui* September!2017! ! ! ! Declaration* I,!Leila!Mekkaoui,!confirm!that!the!work!presented!in!this!thesis!is!my!own.! Where!information!has!been!derived!from!other!sources,!I!confirm!that!this!has! been!indicated!in!the!thesis.! * * * * * * * * * * * * * * Abstract** Lentiviral!vectors!(LVs)!are!powerful!tools!in!gene!therapy!that!have!recently! witnessed!an!increasing!demand!in!both!research!and!clinical!applications.! Current!LVs!purification!represents!the!main!bottle!neck!in!their!application!as! several!methods!are!employed!which!are!time!consuming,!cumbersome!and! yield!low!recoveries.!The!aim!of!this!project!was!to!develop!a!oneMstep!method! to!specifically!and!efficiently!purify!LVs,!with!high!vector!yields!and!reduced! levels!of!impurities,!using!the!biotinMstreptavidin!system.!! Herein,! packaging! 293T! cells! were! genetically! engineered! with! biotin! mimicking!synthetic!peptides!and!different!cell!membrane!anchoring!strategies! for! optimal! streptavidin! binding! were! tested.! We! have! identified! a! flanked! disulphideMconstrained! peptide,! termed! Ctag! (ECHPQGPPCIEGRK),! displayed!on!a!CD8α!stalk!to!be!the!most!promising.!LVs!were!modified!with! Ctag!by!its!random!incorporation!onto!viral!surfaces!during!budding,!without! viral!protein!engineering!or!hindrance!on!infectivity.!The!expression!of!Ctag!on! LVs!allowed!complete!capture!of!infectious!particles!by!streptavidin!magnetic! beads.!As!Ctag!binds!streptavidin!in!the!nanomolar!range,!we!hypothesised! that!gentle!elution!from!streptavidin!matriX!should!occur!by!biotin’s!competitive! binding.! Accordingly,! addition! of! micromolar! concentrations! of! biotin! to! captured! LVs! resulted! in! an! overall! yield! of! ≥60%.! Analysis! of! eluted! LVs! revealed! high! purity! levels,! with! a! ≤3Mlog! and! 2Mlog! reduction! of! DNA! contamination!and!host!cell!proteins,!respectively.!This!oneMstep!purification! was! also! tested! for! scalable! vector! processing! using! streptavidin! monolith! affinity!chromatography!and!preliminary!results!were!encouraging!with!20%! overall!yield.! In! conclusion,! we! developed! a! singleMstep! affinity! chromatography! which! allows! specific! purification! and! concentration! of! infectious! vectors! modified! with!a!biotin!mimic.!Based!on!intended!usage,!efficient!LV!purification!can!be! achieved! using! both! magnetic! beads! and! column! chromatography.! This! method!will!be!of!valuable!use!for!both!research!and!clinical!applications!of! LVs.!! Acknowledgments** I!am!truly!grateful!to!my!supervisors,!Dr.!Martin!Pule,!Dr.!Yasu!Takeuchi!and! Dr.! Ekaterini! (Nina)! Kotsopoulou.! Martin’s! unwavering! support! since! I! first! joined! the! lab,! along! with! his! direction! and! insight! have! been! vital! for! the! success!of!this!project!and!my!focus!throughout!this!PhD.!Yasu’s!continuous! guidance,!rigorousness!and!thoroughness!over!the!years!were!invaluable!for! the!completion!of!this!project.!And!finally,!Nina’s!constant!support,!insight!and! encouragement! throughout! this! work! were! crucial! for! the! direction! of! this! project,!as!well!as!my!sanity.!! I!was!lucky!to!be!part!of!the!Pule!Lab!with!all!its!past!and!present!members! who!have!made!this!eXperience!a!truly!unforgettable!one:!Gordon!WengMKit! Cheung,!Eva!Kokalaki,!Farhan!Parekh,!Brian!Philip,!Shimobi!Onuoha,!Patrycja! Wawryzyniecka,! Cassandra! Stowe,! Paul! Maciocia,! Claire! Roddie,! Guilia! Agliardi,! Lydia! Lee,! Francesco! Nannini,! Ana! Margerida! Pinto! De! Campos,! Alastair!Hotblack,!Annalisa!Maggio,!Vedika!Mehra,!Farhana!Hussain,!Simon! Thomas!and!Shaun!Cordoba.*I!am!sincerely!thankful!for!all!their!help,!advice! and! mostly! for! filling! the! last! 6! years! with! stimulating,! thoughMprovoking,! laughable,!wonderful!and!weird,!with!the!occasional!downMright!unreal!times.! Also!for!humoring!my!neverMending!questions!and!topics!of!conversation!which! went!on!against!their!wills!I’m!aware,!and!to!Gordon!goes!the!biggest.!! I! was! fortunate! enough! to! work! within! the! hematology! department! of! the! Cancer!Institute!that!is!filled!with!kindest!researchers!that!have!never!failed!to! offer! a! helping! hand! both! scientifically! and! personallyf! with! a! very! special! thanks!to!Eva,!Sara!Caxaria,!Solange!Paredes!and!Micaela!Harrasserf!your! friendship!and!support!over!the!years!have!made!this!process!infinitely!lighter.!! I!am!grateful!for!Prof!Farzin!Farzaneh!and!his!team!including!Dr.!David!Darling! and!Dr.!Glenda!Dickson!whose!experience,!advice,!input!and!friendship!were! fundamental!for!the!completion!of!this!project.!! I!am!also!very!thankful!for!the!Takeuchi!Lab’s!collaboration!at!NIBSC!for!their! expertise!and!kind!help!with!various!equipment.!Thanks!to!Dr.!Giada!Mattiuzzo! and!James!Ashall!for!their!advice!and!for!the!Nanosight!experimentsf!and!Dr.! MacLellanMGibson!for!the!introduction!and!the!use!of!electron!microscopy.!! My!thanks!are!also!owed!to!my!friends!outside!of!science!who!have!navigated! the!volatile!journey!of!my!PhD!with!me.!Their!unfailing!support!and!constant! encouragement,!even!in!the!face!of!true!pessimism,!was!indispensable!for!me! throughout!this!period.!! Last!but!not!least,!I!would!like!to!thank!my!family.!I!know!I!don’t!show!it!as! much!as!I!should!but!I!am!beyond!grateful!for!everything!they!have!done!for! me!to!allow!me!to!get!here.!My!biggest!and!warmest!thanks!to!my!parents’! unwavering! support! and! encouragementf! and! to! my! brothers’! abilities! to! lighten!up!any!challenge.!! And!finally,!to!my!partner!Hamoudi,!who!has!trekked!this!journey!with!me!from! the!very!startf!thanks!for!providing!me!endless!inspiration!and!resilience!that! have!eased!this!process!and!for!constantly!motivating!me!when!I!needed!it!the! most. Table*of*contents! DECLARATION*.......................................................................................................*2! ABSTRACT*..............................................................................................................*3! ACKNOWLEDGMENTS*...........................................................................................*4! LIST*OF*FIGURES*.................................................................................................*13! LIST*OF*TABLES*..................................................................................................*15! ABBREVIATIONS*..................................................................................................*16! THESIS*STRUCTURE............................................................................................*18! CHAPTER*1*INTRODUCTION*...............................................................................*19! 1.1! GENE*THERAPY*...............................................................................................*19! 1.1.1! AN!OVERVIEW!OF!GENE!THERAPY!...................................................................!19! 1.1.2! CURRENT!STATUS!OF!GENE!THERAPY!TRIALS!..................................................!21! 1.1.3! FUTURE!CHALLENGES!....................................................................................!25! 1.2! LENTIVIRAL*VECTOR*FOR*GENE*THERAPY*..........................................................*26! 1.2.1! GENERAL!CHARACTERISTICS!AND!BIOLOGY!OF!LENTIVIRUSES...........................!27! 1.2.1.1! HIVM1!architecture:!One!RNA!and!fourteen!proteins..................................!28! 1.2.1.2! HIVM1!life!cycle!..........................................................................................!29! 1.2.2! LENTIVIRAL!VECTOR!DEVELOPMENT!AND!CURRENT!APPLICATIONS!....................!35! 1.3! MANUFACTURING*OF*LENTIVIRAL*VECTORS*.......................................................*42! 1.3.1! LENTIVIRAL!VECTOR!PSEUDOTYPING!...............................................................!42! 1.3.2! LENTIVIRAL!VECTOR!STABILITY!DURING!BIOPROCESSING!..................................!44! 1.3.3! UPSTREAM!PROCESSING!STRATEGIES!............................................................!47! 1.3.3.1! Transient!transfection!...............................................................................!47! 1.3.3.1.1! Adherent!cell!lines!..................................................................................!47! 1.3.3.1.2! Suspension!cells!....................................................................................!49! 1.3.3.2! Packaging!and!producer!cell!lines!.............................................................!50! 1.3.4! DOWNSTREAM!PROCESSING!STRATEGIES!.......................................................!55! 1.3.4.1! Compositions!of!vector!products!...............................................................!56! 1.3.4.2! Overview!on!clinical!grade!downstream!processes!...................................!57! 1.3.4.3! Clarification!...............................................................................................!60! 1.3.4.4! Concentration!...........................................................................................!60! ! 6! ! ! ! 1.3.4.4.1! Centrifugation!.........................................................................................!61! 1.3.4.4.2! Ultrafiltration!...........................................................................................!62! 1.3.4.5! Purification!................................................................................................!62!
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