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`
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`NATIONAL
`NATIONAL
`|IBRARY OF
`LMEDICNE]«=WEDICINE Sie
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`AAV-based gene therapy prevents neuropathology and results in normalcognitive
`developmentin the hyperargininemic mouse
`si
`encipiaainiath ;
`GENE THERAPY
`
`N [2013 VOLUME 2@ ISSUE 8
`
`| (aN0:8;
`
`Page 1 of 12
`
`KELONIA EXHIBIT 1007
`
`Gene therapy.
`v. 20, no. 8 (Aug. 2013)
`General Collection
`W1 GE184Q
`2013-08-28 13:22:13
`
`Volume 20 Number8
`
`August 2013
`
`www.nature.com/gt
`
`7
`
`a.A
`
`ACMeteatte
`
`ic vehicle for CNS
`
`En
`
`ature publishing
`P
`
`grou
`6 5f0up
`
`5
`
`IND
`SR96801
`
`Page 1 of 12
`
`KELONIA EXHIBIT 1007
`
`

`

`Gene Therapy
`
`European Editor
`Nicholas Lemoine
`London, UK
`
`USA Editor
`Joseph Glorioso
`Pittsburgh, PA, USA
`
`Associate Editors
`
`Robin Ali
`University College London, UK
`Roger Hajjar
`Mount Sinai School of Medicine, New York, NY, USA
`Steven Ghivizzani
`University of Florida, Gainesville, FL, USA
`Antonio Chiocca
`Ohio University, Columbus, OH, USA
`
`David Fink
`University of Michigan, Ann Arbor, Ml, USA
`Edvard Smith
`Karolinska Institute, Stockholm, Sweden
`Xuetao Cao
`Second Military Medical University, Shanghai, P. R. China
`
`Editorial Office
`
`Virginia Boylan
`
`Aims and Scope
`
`Gene Therapy covers both the research and clinical applications of the new genetic therapy techniques currently being
`developed. Over the last decade, gene therapy protocols have entered clinical trials in increasing numbers and as they
`cover a wide spectrum of diseases, these studies promise to unite the diverse organ-based specialities into which
`modern medicine has becomedivided. Gene Therapy coversall aspects of gene therapy as applied to human disease,
`including:
`
`Novel technological developments for gene transfer, control and silencing
`Basic science studies of mechanisms of gene transfer and control of expression
`Preclinical animal model systems and validation studies
`Clinical trial reports which have significant impact for the field
`Gene-based vaccine development and applications
`Cell-based therapies including all aspects of stem cells and genetically modified cellular approaches
`
`Page 2 of 12
`
`Page 2 of 12
`
`

`

`Gene Therapy
`
`
`
`August 2013
`
`
`CONTENTS
`
`Volume 20, Number 8
`
`| ORIGINAL ARTICLES
`
`785
`
`797
`
`807
`
`816
`
`AAV-based gene therapy prevents neuropathology and results in normal cognitive development in the hyperargininemic
`mouse
`EK Lee, C Hu, R Bhargava, R Ponnusamy, H Park, S Novicoff, N Rozengurt, B Marescau, P De Deyn, D Stout, L Schlichting,
`WW Grody, SD Cederbaum and GS Lipshutz
`
`Engineered stem cell-derived microglia as therapeutic vehicle for experimental autoimmune encephalomyelitis
`C Beutner, V Lepperhof, A Dann, B Linnartz-Gerlach, S Litwak, | Napoli, M Prinz and H Neumann
`
`Engineering a serum-resistant and thermostable vesicular stomatitis virus G glycoprotein for pseudotyping retroviral
`and lentiviral vectors
`B-Y Hwang and DV Schaffer
`
`Novelelectric power-driven hydrodynamic injection system for gene delivery: safety and efficacy of human
`factor IX delivery in rats
`T Yokoo, K Kamimura, T Suda, T Kanefuji, M Oda, G Zhang, D Liu and Y Aoyagi
`
`824
`
`Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus
`VS Lopes, SE Boye, CM Louie, $ Boye, F Dyka, V Chiodo, H Fofo, WW Hauswirth and DS Williams
`
`834
`
`846
`
`Suppression of breast tumor growth by DNA vaccination against phosphatase of regenerating liver 3
`J Lv,
`Liu, H Huang, L Meng, B Jiang, Y Cao, Z Zhou, T She, L Qu, S Wei Song and C Shou
`
`Hydrodynamic delivery of adiponectin and adiponectin receptor 2 gene blocks high-fat diet-induced obesity
`andinsulin resistance
`Y Ma and D Liu
`
`SHORT COMMUNICATIONS
`
`| 853
`
`Efficient selection of genetically modified human T cells using methotrexate-resistant human dihydrofolate
`reductase
`M Jonnalagadda, CE Brown, WC Chang, JR Ostberg, SJ Forman and MC Jensen
`
`861
`
`868
`
`Multi-cistronic vector encoding optimized safety switch for adoptive therapy with T-cell receptor-modified T cells
`MM van Loenen, R de Boer, RS Hagedoorn, V Jankipersadsing, AL Amir, JHF Falkenburg and MHM Heemskerk
`
`Lack of genotoxicity due to foamy virus vector integration in human iPSCs
`DR Deyle,
`IF Khan, G Ren and DW Russell
`
`@
`
`nature publishing group
`
`
`
`Copyright © 2013 Macmillan Publishers Limited
`
`Subscribing organisations are encouraged to copy and distribute
`this table of contents for internal, non-commercial purposes
`
`This issue is now available at:
`www.nature.com/gt
`This journal is a member of, and subscribes to the principles of, the
`Committee on Publication Ethics (COPE) www.publicationethics.org
`
`cor |
`
`
`
`Page 3 of 12
`
`Page 3 of 12
`
`€
`

`

`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Page 4 of 12
`
`

`

`residues (Thr344, lle98, Phe125 and Phe470) would appear unable
`to interact with components of human serum, and the former set
`of mutations was instead investigated.
`
`Functional analysis of individual VSV-G mutations
`As there were no dominant individual clones following selection,
`we analyzed the roles of the common mutations in potential VSV-G
`serum resistance. Site-directed mutagenesis was used to introduce
`the individual, identified point mutations into the surface-exposed
`positions of wild-type VSV-G (Figure 1), and GFP-expressing retroviral
`vectors were packaged. We found that with the exception of $162T,
`the genomic titers (G-titers) of mutant vectors were equivalent to
`wild-type vector (Figure 2a), suggesting that VSV-G can tolerate
`mutations that emerged from this library selection without the loss
`of assembly. The mutantinfectioustiters (\-titers) were similar to wild
`type, with the exceptions that VSV-G E380K exhibited a 40%
`increase, and K66T and T368A showed a 70% decrease,respectively,
`in infectivity (Figure 2b). We investigated the relativeratio of I-titer to
`G-titer of all mutant variants (Figure 2c and Supplementary Data
`Figure S4C). Vector containing K66T, $162T, T230N or T368A VSV-G
`showed somewhatlower I-titer/G-titer ratios compared with the
`wild type, suggesting that these mutations slightly decreased the
`infectivities of the viral vectors. We also investigated the relative
`ratio of VSV-G expression level, as obtained with a VSV-G Enzyme
`linked immunosorbent assay (ELISA), to G-titer (Figure 2c), and did
`not observe any significant differences compared with wild-type
`VSV-G.
`
`Engineering a serum-resistant and thermostable VSV-G
`B-Y Hwang and DVSchaffer
`
`e 8
`
`08
`
`sensitivity to human complement, through this approach has not
`been broadly explored.”°
`Directed evolution has recently been developed and imple-
`mented to improve numerous properties of viral vectors, and this
`approach can be effective even in the absence of a mechanistic
`understanding of challenges facing a vector system.In particular,
`multiple studies have applied molecular evolution to improve
`vector stability, pseudotyping efficiency, transduction efficiency,
`resistance to antibodies, genomic integration selectivity and other
`properties.**** Here, we explore whether directed evolution of
`the VSV-G envelope may enable pseudotyped viral vectors to
`resist neutralization by human serum. Through a combination of
`evolution and site-directed mutagenesis, we created VSV-G
`variants that are both resistant to a panel of human and animal
`sera and are thermostable. Furthermore, variants exhibited enhan-
`ced gene delivery in the presence of human serum in vivo.
`
`RESULTS
`
`Library construction and selection
`two
`VSV-G libraries were constructed by error-prone PCR at
`different mutation rates: a 210° mutant
`library with a low
`error
`rate of 1-4 nucleotide mutations per VSV-G sequence
`and a 2x 10° mutant
`library with 3-8 nucleotide mutations
`per sequence, as quantified by sequencing of randomly chosen
`clones. For selection, these libraries were inserted into a retroviral
`vector plasmid that was used to package a library of vector
`particles, where each harbored a vector genome encoding the
`Human serum inactivation of variant VSV-G retroviral vectors
`VSV-G variant incorporated in the envelope of that particle. This
`The human serum inactivation of retroviral vectors bearing wild
`requires that ~ 1 plasmid carrying a VSV-G variant be transfected
`type and single mutant VSV-G variants was examined. As
`into a producer cell during packaging. Forinitial optimization of
`noninfectious of empty particles could conceivably affect a
`this process, Human Embryonic Kidney (HEK) 293T cells were
`neutralization assay, we used the results of a VSV-G ELISA assay
`transfected with 62.5 ng—2 wg of the retroviral vector pCLPIT GFP
`(Supplementary Data Figure $4C) to equalize the amountof VSV-G
`followed by flow cytometry analysis (Supplementary Data Figure
`added to each sample. The resulting ELISA-normalized viral
`$1). Upon transfection with 62.5 ng of pCLPIT GFP, ~ 15% of cells
`vectors levels were incubated with the mixture of human serum
`expressed GFP, suggesting that these conditions may introduce
`from 18 donors. Following serum incubation at 37Cfor 1h, titers
`on average <1 plasmid per cell and could thus produce the
`were quantitated and reported as the percentage of remaining
`desired virion library. Therefore,
`the two CLPIT VSV-G libraries
`titer compared with that of control samples incubated at 37 C for
`were packaged separately using these conditions, and the
`1h with phosphate-buffered saline (PBS) (pH 7.4) (Figure 3a) or
`resulting vectors were combinedfor selection.
`heat inactivated (56 C, 30min) human sera (HIHS) (Supplemen-
`To develop a strategy for selecting VSV-G variants resistant to
`tary Data Figure $5A). Viral vectors were not inactivated in HIHS,
`serum neutralization, retroviral vector pseudotyped with wild-type
`suggesting that the inactivation may be caused by complement
`VSV-G was diluted fivefold in a mixture of human serum from 18
`(Supplementary Data Figure S5B), and the results of these two
`donors and incubated at 37 C (Supplementary Data Figure $2).
`normalization were thus very comparable.In particular, individual
`Vector infectivity progressively decreased with longerincubations,
`VSV-G mutants $162T (P<0.05), T230N (P<0.05) or T368A
`such that only ~ 5%of the vector remainedinfective after 6h. The
`(P<0.05) showed significantly increased human serum resistance
`VSV-G library was
`thus
`selected to evade human serum
`compared with wild-type VSV-G (Figure 3a).
`inactivation by incubating the virions with serum at 37 C for
`6h. 293T cells were then infected with the treated viral vector
`Incubation in serum at 37 C may select not only for serum
`resistance but serendipitously also for
`thermostability, so we
`library (at a multiplicity of infection<0,1 to prevent multiple
`further examined the results for the vectors before and after
`infections of a single cell). Following the selection with 1 jig ml |
`incubation for 1h in PBS (Figure 3b). Mutants carrying K66T, T368A
`of puromycin, the selected viral pool was rescued via transfection
`or E380K interestingly showed higher thermostability compared
`of the pCMV gag/pol helper plasmid.
`with wild-type VSV-G (P<0.05). Because of the combined effects
`This process was repeated for six selection steps, and VSV-G
`of serum resistance and thermal stability, a mutant carrying T368A
`variants were then recovered from 293T cell genomic DNA via
`showed > 1.6-fold higher infectivity (P<0.01) compared with wild-
`PCR and inserted into the plasmid pcDNA IVS to generate
`type VSV-G upon incubation in human serum at 37°C for 1h
`helper plasmids for vector production. DNA sequencing revealed
`(Figure 3c). On the basis of these data, the $162T, T230N and
`that while there were no duplicates among 36 randomly chosen
`T368A mutations were deemed beneficial for serum resistance.
`VSV-G clones, common mutations were present
`(Figure 1a;
`Combining mutations that individually contribute to a given
`Supplementary Data Figure $3). The positions of these ‘hot spot’
`property can further enhance that property.*'“* We therefore,
`mutations were analyzed within the structure of the prefusion
`generated a number of double and triple VSV-G mutants from
`form of VSV-G (Figure 1b),“° which indicated that Lys66, Ser162,
`individual mutations shown to confer serum resistance. All VSV-G
`Asp208, Ser212, Lys216, Thr230, Thr368 and Glu380 are located on
`mutants analyzed showedsignificantly higher resistance (P<0.01
`the protein surface.
`In addition, Thr344 is
`located in
`the
`or 0.05) to human serum (gray bars in Figure 4a). Furthermore, all
`core region, lle98 and Phe125 are located on the leg region that
`such mutants showed similar thermal stability compared with
`points toward the viral membrane and Phe470 is located on the
`wild-type VSV-G (striped bars in Figure 4a). For comparison, we
`transmembrane domain of VSV-G. The latter, nonsolvent exposed
`
`Gene Therapy (2013) 807-815
`
`2013 Macmillan Publishers Limited
`
`Page 5 of 12
`
`Page 5 of 12
`
`€
`

`

`Engineering a serum-resistant and thermostable VSV-G
`B-Y Hwang and DV Schaffer
`
`809
`
`
`
`a 66
`
`> 0.5
`=]
`55
`04
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`©
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`= 0.1
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`301
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`351
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`401
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`451
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`504
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`E380K
`S212E
`T368A
`
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`T230N/A
`T3444
`S
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`F4708
`
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`/
`201 251
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`154
`
`Position
`
`Figure 1. Common mutations following directed evolution of VSV-G. (a) The frequency of mutation at each amino acid residue of VSV-G
`among 36 randomly chosen VSV-G clonesafter five or six selection steps. (b) The location of each apparent ‘hot spot mutation’in the crystal
`structure of the prefusion form of VSV-G (PDBID: 2J6J). Figure was made using PyMol (http://www.pymol.org). Each monomer of VSV-G was
`colored in green, purple and sky blue, respectively. Green, blue and red balls represent carbon, nitrogen and oxygen atoms, respectively.
`
`also prepared retroviral vector pseudotyped with an RD114
`envelope, which has previously been reported to have higher
`serum resistance than wild-type VSV-G,**" as another control. As
`reported, RD114 showed increased resistance to human serum
`compared with wild-type VSV-G,
`though again this envelope
`cannot package vector to the high titers needed for in vivo use.**
`Collectively,
`the T230N+7T368A mutant
`showed the highest
`residual infectivity (~ 68%) compared with the residual infectivity
`of parental VSV-G (~ 37%) and similar residual infectivity (~ 68%)
`of RD114 after incubation in human serum at 37 C for 1h.
`K66T, T368A and E380K mutations increased VSV-G thermal
`Stability (Figure 3), and we thus investigated the effects of adding
`these mutations
`to the promising serum-resistant variants.
`Several variants containing K66T or K66T + E380K substitutions
`showed similar or slightly higher thermal stability compared
`with wild-type VSV-G, and several mutants
`(K66T +S162T +
`T230N+7368A, K66T+T368A+E380K and K66T+S$162T+
`T230N + E380K) also showedstatistically higher serum resistance
`(P<0.05) (Figures 4b andc).
`
`Human serum inactivation of variant VSV-G lentiviral vectors
`Although the envelope compositions of retroviral and lentiviral
`vectors are likely similar, we also analyzed the ability of VSV-G
`
`variants to confer serum resistance to lentivirus. On the basis of
`results with retroviral vectors, we analyzed the top five VSV-G
`variants (S162T + T230N, S162T + T368A, T230N + T368A, K66T +
`T368A+E380K and K66T+5$162T +1T230N+T368A)
`showing
`higher
`serum resistance
`for
`retrovirus. Equal
`amounts of
`packaged lentivirus (7 x 10° GFP TU) were diluted fivefold in
`human serum, or PBS (pH 7.4) as a control, and incubated at 37 C
`for 1h. Vector pseudotyped with several mutant VSV-G showed a
`greater resistance to human serum compared with wild-type
`VSV-G pseudotyped lentiviral vector
`(Figure 5).
`Importantly,
`mutants
`1T230N+7368A or K66T+5S162T+7230N+1368A
`had higher combined thermostability and serum resistance than
`wild-type VSV-G.
`
`Vector inactivation by animal serum
`VSV-G pseudotyped viral vectors can be inactivated by sera from
`mouse, rat and guinea pig, which can impact the interpretation of
`animal studies.”’“* The relative sensitivity of VSV-G pseudotyped
`retroviral or lentiviral vectors to several animal sera was thus
`tested (Figures 6a and b, respectively). $162T +T230N, T230N +
`T368A or K66T + $162T + T230N + T368A VSV-G showed statisti-
`cally higher resistance to mouse and rabbit sera for both retroviral
`and lentiviral vectors.
`
`© 2013 Macmillan Publishers Limited
`
`Gene Therapy (2013) 807-815
`
`me
`Page 6 of 12
`
`Page 6 of 12
`
`

`

`¢
`810
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`Engineering a serum-resistant and thermostable VSV-G
`B-Y Hwang and DV Schaffer
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`Figure 2. Genomic andinfectioustiters of VSV-G chimeric retroviral
`vectors. Murine retroviral vector was packaged with the pCLPIT GFP
`vector plasmid, pCMV gag-pol and pcDNAIVS VSV-G helper plasmid
`containing individual VSV-G variants.
`(a) Vector genomic titers
`(G-titers) were measured by real-time quantitative PCR. (b) Trans-
`duction efficiencies on 293T cells were determined by flow
`cytometry analysis of retroviral vector mediated GFP expression.
`(c) Relative ratios of infectioustiters (Ititers) to G-titers (gray bars)
`and VSV-G ELISA data to G-titers (black bars) of the retroviral vectors
`were calculated with the ratio of wild-type VSV-G pseudotyped
`retroviral vector as 1, respectively. Error bars denote s.d. (n= 3).
`* and ** indicate statistical differences of P<0.05 and P<0.01,
`respectively, compared with infectivity of wild-type VSV-G,
`as
`determined using an one-way analysis of variance (ANOVA).
`
`b 100.090.0
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`Figure 3, Human serum resistance and thermostability of retroviral
`vectors pseudotyped with VSV-G mutants. The amounts of viral
`vectors were normalized based on VSV-G ELISA assay. (a) Human
`serum neutralization was quantified by measuring vectortiters after
`incubation with human serum at 37 C for 1h,relative to those after
`incubation with PBS at 37 C for 1h.
`(b) Thermal effects were
`determined by quantifying relative titers after incubation with PBS
`at 37 C for 1h compared with those without incubation at 37 C.
`(c) Human serum neutralization and thermal effects were deter-
`mined by calculation of relative titers after incubation with human
`serum at 37 C for 1h compared with those without incubation at
`37 C. Error bars denote s.d. (n=4). * and ** indicate statistical
`differences of P<0.05 and P<0.01, respectively, compared with
`infectivity of wild-type VSV-G, as determined using an one-way
`ANOVA.
`
`In vitro transduction of several cell lines
`Although theinfectivities of 293T cells were comparable with wild-
`type VSV-G in the absence of human serum (Figure 2), we
`characterized transduction of several other cell lines to determine
`whether the mutations affected infectivity (Figure 7). Although the
`infectivities of the variants were somewhat lower for several cell
`types, the trends in infectivity for all
`lines was similar between
`wild-type and mutant VSV-G. Interestingly, the mutant carrying
`K66T + $1621 +1230N +1368A showedstatistically higher trans-
`duction efficiency to Hela cells compared with wild type
`(P<0.01).
`
`In vivo analysis of human serum resistance
`The ability of two promising variants—which we now term mutant
`1
`(T230N+7368A)
`and mutant
`2
`(K66T+$162T+T230N+
`T368A)—to resist human serum neutralization in
`a murine
`model was analyzed. For the in vivo neutralization assays, vector
`could be added to 100% human serum; however, human serum
`
`administration in vivo results in an ~ 10-fold dilution of the serum
`into circulation. To tune the level of vector that should be
`administered to observe neutralization, a preliminary study with
`wild-type VSV-G was conducted, and the interval between serum
`and lentiviral vector administration was also varied. BALB/c mice
`were injected with 20041 of human serum, or PBS as a control,
`into the tail vein. One, 5, or 24h later, lentiviral vector encoding
`firefly luciferase was administered. For an 8 x 10'° vector genome
`administration, after two weeks expression was observed in the
`liver,
`the primary site of lentiviral vector transduction.” Also,
`expression levels were lower for vector administered 1 or 5h, but
`not 24h,after infection of human serum, compared with the PBS
`control (data not shown).
`To assess neutralization of the VSV-G mutants, equal levels of
`lentiviral vector (8 x 10'° vector genomes) were injected via the
`tail vein 1h after the administration of 200 ul of human serum of
`PBS. After 2 weeks, the mice were killed, and luciferase expression
`levels were analyzed in the liver. The presence of human serum
`reduced luciferase expression mediated by wild-type VSV-G to
`
`Gene Therapy (2013) 807-815
`
`Page 7 of 12
`
`© 2013 Macmillan Publishers Limited
`
`
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`Page 7 of 12
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`

`

`Engineering a serum-resistant and thermostable VSV-G
`
`B-Y Hwang and DV Schaffer
`
`0.0 Residual
`
`Infectivity(%)
`
`\N
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`NN
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`T230N,
`T3684,
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`T230N
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`
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`T368A,
`E380K
`
`K66T,
`$162T,
`T230N,
`T368A
`
`5. Human serum neutralization and thermostability of
`Figure
`lentiviral vectors pseudotyped with VSV-G variants. A standard
`GFP encoding lentiviral vector was packaged with five VSV-G
`mutants that appeared promising in the retroviral results. Five VSV-G
`variants ($162T+T230N, S162T + T368A, T230N+7368A, K66T +
`T368A + E380K and K66T + $162T + T230N + T368A) showing higher
`resistance and thermal stability for retroviral vector packaging were
`selected. Thermal effects, human serum neutralization and com-
`bined serum neutralization and thermal effects were determined by
`quantifying relative titers after incubation with PBS at 37°C for 1h
`compared with those without incubation at 37°C, after incubation
`with human serum at 37 C for 1h compared with those after
`incubation with PBS at 37°C for 1h, and after incubation with
`human serum at 37 C for 1h compared with those without
`incubation at 37 C,
`respectively. Error bars denote s.d. (n= 4).
`* and ** indicate statistical differences of P<0.05 and P<0.01,
`respectively, compared with the wild-type VSV-G, as determined
`using an one-way ANOVA.
`
`
`
`RDW14
`
`WT
`
`$1627, T230N S162T,T36BA T230N,T368A $4162T,
`
`T230N,A
`
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`rmal
`120.0
`a 420>|= Thermal
`Serum
`Serum
`mal+Serum
`4
`@ Thermai+Serum!
`
`100 +
`SS
`
`
`
`ResidualInfectivity(%)
`(%) RDIW14
`
`ResidualInfectivity
`
`
`
`WT
`
`
`
`vehicles for gene transfer gene in vitro and in vivo. However,
`while VSV-G pseudotyped vectors
`have been increasingly
`explored for ex vivo gene delivery in clinical
`trials,#°*” serum
`neutralization of
`the VSV-G pseudotyped viral vectors
`is
`a
`significant obstacle for direct
`in vivo administration.
`In this
`study, we conducted directed evolution of VSV-G to create
`K66T,
`K667,
`K66T,
`K66T,
`Ké66T,
`K66T,
`K66T,
`$1627,
`T230N,
`$1627,
`$162T,
`368A,
`T230N,
`S162T,
`serum-resistant VSV-G mutants. After six selection steps, followed
`T230N,
`T36BA,
`T368A,
`T230N,
`E380K
`E380K
`E380K
`
`
`E3s0K §=6E380K=T368A,«6E380K
`by functional analysis of common mutations, we identified several
`E380K
`mutations ($162T, T230N and T368A) that enhanced VSV-G serum
`resistance. Furthermore, as incubation at 37 C was inherent to
`the selection, we also identified several adventitious mutations
`(K66T, T368A and E380K)
`that apparently increased VSV-G
`thermostability. Upon combining these two classes of mutations,
`retroviral and lentiviral vectors pseudotyped with two resulting
`VSV-G_
`variants
`(T230N+T368A and K66T+S$162T+7230N
`+ 1368A) showed higher resistance to human and animal sera,
`as well as increased thermostability compared with wild-type
`VSV-G. While adeno-associated virus has been evolved to resist
`antibody neutralization,*®***? to our knowledge this is the first
`effort to harness iterative random mutagenesis and selection to
`endow retroviral or lentiviral vectors with the capacity to resist
`serum inactivation.
`VSV-G pseudotyped viral vectors can be inactivated in serum by
`complement,“°*° a major elementof the innate immune response
`that
`in general
`functions via classical, alternative and lectin
`pathways.”' All pathways can mediate virus opsonization, virolysis
`and anaphylatoxin and chemotaxin production. However,
`the
`particular mechanisms by which complement inactivates VSV-G
`presenting virions are unknown. The mutation sites
`(Ser162,
`Thr230 and Thr368) we identified do notlie within several known
`antibody binding epitopes,”° indicating that these residues may
`lie in previously uncharacterized epitopes, or that VSV-G may
`interact with complement via mechanisms
`independent of
`antibody
`neutralization. Despite
`the
`lack of mechanistic
`information underlying a given gene delivery problem, directed
`evolution can still be used to create enhanced variants, which in
`
`4. Human serum neutralization and thermostability of
`Figure
`retroviral vectors pseudotyped with VSV-G variants that combine
`several
`‘hot
`spot mutations! VSV-G mutants with combined
`beneficial mutations were generated bysite-directed mutagenesis.
`The amounts ofviral vectors were normalized based on VSV-G ELISA
`assay. Thermal effects, human serum neutralization and combined
`serum neutralization and thermal effects were determined by
`quantifying relative titers after incubation with PBS at 37 C for 1h
`compared with those without incubation at 37 ‘C, after incubation
`with human serum at 37 C for 1h compared with those after
`incubation with PBS at 37°C for 1h, and after incubation with
`human serum at 37°C for 1h compared with those without
`incubation at 37 C, respectively. (a) Variants contained ‘hot spot
`Mutations’ for human serum resistance. (b) K66T and (¢) E380K were
`added to enhance the thermal stability of retroviral vectors. Error
`bars denote s.d. (n=4). * and ** indicate statistical differences
`of P<0.05 and P<0.01, respectively, compared with the wild-type
`VSV-G, as determined using an one-way ANOVA.
`
`Only 22.1% of PBS-injected controls in the liver. The human serum
`reduced mutants 1 and 2 to only 60%of PBS controls, levels that
`Were nearly threefold higher than wild-type VSV-G under the same
`Conditions (Figure 8).
`
`DISCUSSION
`their broad tropism and high stability, VSV-G
`Because of
`Pseudotyped retroviral and lentiviral vectors are promising
`
`© 2013 Macmillan Publishers Limited
`
`
`Page 8 of 12
`
`Gene Therapy (2013) 807-815
`
`Page 8 of 12
`
`

`

`S WT
`G T230N, T368A
`@ K66T, $162T, T230N, T368A
`
`
`
`
`
`Relativeluciferaseactivity(%)
`
`
`
`
`
`two variants
`Figure 8. Human serum neutralization in vivo. For
`(T230N +7368A and K66T + $162T + T230N + T368A) and wild type
`VSV-G,lentiviral vectors encoding luciferase were administered via
`tail vein injection to female BALB/c mice one hour after human
`serum or PBS introduction. After two weeks,
`levels of luciferase
`activity were determined and normalized to total protein for each
`sample analyzed. Relative
`luciferase
`expression in
`liver was
`determined by quantifying relative enzyme activity from human
`serum-primed mice relative to activity from naive mice. Error bars
`denote s.d. (n = 4). * and ** indicate statistical differences of P<0.05
`and P<0.01,
`respectively, compared with wild type VSV-G, as
`determined using an one-way ANOVA.
`
`Engineering a serum-resistant and thermostable VSV-G
`B-Y Hwang and DVSchaffer
`
`aWT
`(0 $162T, T230N
`5 T230N, T368A
`©) K66T, $1627, T230N, T368A
`
`Mouse
`
`Rabbit
`
`a
`
`=
`=&
`eao
`
`=a33w®
`
`iv
`
`5@e N
`
`b 100.0 5
`
`= 300 |
`
`
`
`
`
`
`60.0
`
`4005
`
`20.0 4
`
`0.0
`
`Mouse
`
`Rabbit
`
`2==
`
`33a o
`
`e
`
`2&w
`
`e
`
`Figure 6. Aminal serum neutralization of retroviral and lentiviral
`vectors pseudotyped with VSV-G variants. For
`three variants
`(S162T +T230N, T230N +T368A and K66T+$162T +T230N +
`T368A) and wild-type VSV-G, neutralization by animal sera was
`examined.
`(a) Retroviral vectors and (b)
`lentiviral vectors were
`diluted fivefold in animal sera and incubated at 37 C for 1h, Serum
`inactivation was determined by quantifying relative titers after
`incubation with human serum at 37 C for 1h compared with those
`after incubation with PBS at 37 C for 1h. Error bars denote s.d.
`(n=4). * and ** indicate statistical differences of P<0.05 and
`P<0.01,
`respectively, compared with the wild-type VSV-G,
`as
`determined using an one-way ANOVA.
`
`administration. The extracellular half-life of retroviral vectorslies
`between 3.5 and 8h at 37°C°°°'? and our measurement
`(t1/2= ~3h) is close to this range. Therefore, our incubation of
`the library for 6h in human serum at 37 C also selected for
`enhanced thermostability. An increased protein thermal stability
`can be caused by several factors such as disulfide bond formation,
`hydrophobic interactions or change of electrostatic interactions of
`the surface.*? The E380K mutation in VSV-G may eliminatealike-
`4.0E+08
`charge
`repulsion with Asp381
`and create opposite-charge
`attraction on the protein surface to confer thermostability to the
`protein. By comparison, potential mechanisms for K66T and T368A
`thermostabilization are not as readily apparent. At any rate,
`combining these mutations with others that conferred serum
`resistance resulted in vectors with both the properties.
`In
`particular, mutants 1 and 2 showed a twofold increase in half-
`life at 37 -C (ty. = ~ 6h). These improvements of thermal stability
`may have also contributed slightly to a greater stability to vector
`concentration by ultracentrifugation (data not shown).
`for
`Cocal-pseudotyped lentiviral vectors may have potential
`in vivo gene therapy, due to their high titers, broad tropism,
`stability and reported increased resistance to human serum
`compared with VSV-G pseudotyped vectors.*' However, unlike
`VSV-G,
`their
`in vivo use has not yet been reported. RD114-
`pseudotped vectors also exhibit
`resistance to human serum.
`Green et al. ** conducted immunostaining for RD114 receptors,
`which were present in the colon,testis, bone marrow,skeletal
`muscle and skin epithelia. However, the receptors are absent in
`the lung, thyroid and artery, suggesting that the application of
`RD114-pseudotyped vectors to treat some diseases may be
`limited. By comparison, VSV-G pseudotyped lentiviral vectors
`have potential for broad tropism.”*
`In this study, we evolved VSV-G and successfully created
`variants with higher resistance to human, rabbit and mouse sera
`in vitro and human serum in vivo. This work therefore further
`establishes the power of directed evolution to improve viral
`
`=mm4s 1.0E+05
`
`WT
`
`$162T, T230N
`
`T230N, T368A
`
`
`
`Titer(TU/ml)
`
`lines with retroviral
`/n vitro transduction of multiple cell
`Figure 7.
`vectors pseudotyped with VSV-G variants. Retroviral vectors expres-
`sing GFP were used to transduce a panel of cell
`lines: HEK 293T,
`HT1080 (human fibrosarcoma cell
`line), CHO K1, NIH 3T3 (mouse
`embryonic fibroblast
`cell
`line), and HeLa cells to assess
`the
`transduction profile of the novel VSV-G variants. Error bars denote
`s.d.
`(n=3).
`**
`indicates
`statistical differences of P<0.01,
`as
`determined using an one-way ANOVA,
`
`this case could aid future efforts to investigate mechanisms of
`retroviral or lentiviral vector inactivation by complement.
`As a byproduct of the evolution, mutations that enhanced the
`thermal
`stability of VSV-G were also identified, which can
`potentially enhance vector production and reduce the dosage of
`
`Gene Therapy (2013) 807-815
`
`© 2013 Macmillan Publishers Limited
`
`Page 9 of 12
`
`Page 9 of 12
`
`

`

`vectors, and these results may enhance the utility of retroviral and
`jentiviral vectors to treat human disease. In addition, VSV itself has
`emerged as a promising candidate in the field of oncolytic virus
`therapy. Therefore, incorporating a VSV variant encoding a human
`serum-resistant and thermostable VSV-G may enhance the thera-
`peutic potential of VSV for treating human cancer.
`
`MATERIALS AND METHODS
`Library construction and selection
`Random mutagenesis libraries were generated by standard error-prone
`PCR of a VSV-G template using VSV-G fwd and VSV-G rev as primers (see
`Supplementary Table S1
`for all primer sequences). The amplified PCR
`fragments were digested with Sf | and Xho|, and the resulting fragments
`were ligated into the corresponding sites of the retroviral vector pCLPIT,
`which contains a puromycin resistance gene and places VSV-G under a
`tetracycline responsive promoter.
`HEK 2937 cells were cultured in Iscove’s modified Dulbecco’s medium
`supplemented with 10% fetal bovine serum (Invitrogen, Calrsbad, CA, USA)
`and 1%penicillin/streptomycin (Invitrogen) at 37 C and 5% CO,. The VSV-
`G library was packaged into retroviral vectors via calcium phosphate
`transfection of 62.5ng of pCLPIT