Chapter 5 - Springer Link

Chapter 5 Engineering HSV-1 Vectors for Gene Therapy William F. Goins, Shaohua Huang, Justus B. Cohen, and Joseph C. Glorioso Abstract Virus vectors have been employed as gene transfer vehicles for various preclinical and clinical gene therapy applications, and with the approval of Glybera (alipogene tiparvovec) as the first gene therapy product as a standard medical treatment (Yla-Herttuala, Mol Ther 20: 1831–1832, 2013), gene therapy has reached the status of being a part of standard patient care. Replication-competent herpes simplex virus (HSV) vectors that replicate specifically in actively dividing tumor cells have been used in Phase I–III human trials in patients with glioblastoma multiforme, a fatal form of brain cancer, and in malignant melanoma. In fact, T-VEC (talimogene laherparepvec, formerly known as OncoVex GM-CSF) displayed efficacy in a recent Phase III trial when compared to standard GM-CSF treatment alone (Andtbacka et al. J Clin Oncol 31: sLBA9008, 2013) and may soon become the second FDA-approved gene therapy product used in standard patient care. In addition to the replication-competent oncolytic HSV vectors like T-VEC, replicationdefective HSV vectors have been employed in Phase I–II human trials and have been explored as delivery vehicles for disorders such as pain, neuropathy, and other neurodegenerative conditions. Research during the last decade on the development of HSV vectors has resulted in the engineering of recombinant vectors that are totally replication defective, nontoxic, and capable of long-term transgene expression in neurons. This chapter describes methods for the construction of recombinant genomic HSV vectors based on the HSV-1 replication-defective vector backbones, steps in their purification, and their small-scale production for use in cell culture experiments as well as preclinical animal studies. Key words Herpes simplex virus, Gene therapy, Gene transfer, Virus vectors, Virus purification, Virus production

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Introduction Over the last twenty years gene therapy has made tremendous inroads from transitioning from the development of methodologies to deliver genes to cells, to in vivo delivery of therapeutic genes in various animal disease models, and finally to use in human clinical trials. During the last year, we have now seen the approval of the first gene therapy product (Glybera; alipogene tiprvovec; AAV1-LPLS447X) using adeno-associated virus (AAV) in this instance to express lipoprotein

Russell J. Diefenbach and Cornel Fraefel (eds.), Herpes Simplex Virus: Methods and Protocols, Methods in Molecular Biology, vol. 1144, DOI 10.1007/978-1-4939-0428-0_5, © Springer Science+Business Media New York 2014

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lipase to treat the rare autosomal recessive lipoprotein lipase deficiency [1] following successful Phase-II and III studies. Thus represents the first major success for the field demonstrating that a single, simple intra-muscular injection of the biologic provided sustained therapeutic benefits. This encouraging result was followed by a very successful Phase-I trial to treat stage III/IV malignant melanoma using a replication-competent oncolytic herpes simplex virus (HSV) vector (T-VEC; talimogene laherparepvec; OncoVex GM-CSF) that provided a 26 % response rate [2]. These two gene therapeutics that demonstrated significant clinical benefit have revitalized the field of gene therapy and show that each vector system will be useful in particular clinical niches. Thus, engineering of such vector systems including their production and purification will continue to be important features for their future development and use. Herpes simplex virus (HSV) is one of the eight members of the human herpesvirus family including HSV-1 (HHV-1) and HSV-2 (HHV-2) serotypes, varicella zoster virus (VZV or HHV-3), Epstein–Barr virus (EBV or HHV-4), human cytomegalovirus (HCMV of HHV-5), human herpesvirus-6 (HHV-6), human herpesvirus-7 (HHV-7), and Kaposi’s sarcoma herpes virus (KSHV or HHV-8), all of which cause some form of human disease and are capable of long-term persistence within specific cells of the human host. Of the three neurotropic herpesviruses or alphaherpesviruses (HSV-1, HSV-2, and VZV), HSV-1 contains a 152 kb linear double-stranded DNA genome encoding approximately 85 gene products [3]. The HSV genome (Fig. 1a) is composed of two segments, the unique long (UL) and unique short (US) components, each of which is flanked by inverted repeats containing important viral regulatory genes and elements. With few exceptions, HSV genes are present as contiguous transcription units in a single copy, which make their genetic manipulation relatively straightforward for the construction of recombinant vectors with the exception being the genes present as two identical copies within the inverted repeats. The HSV particle (Fig. 1b) comprises over 34 proteins with an icosahedral shaped nucleocapsid composed of structural capsid proteins surrounded by a lipid envelope bilayer possessing virus-encoded glycoproteins essential for attachment and penetration of the virus into receptor-bearing cells. Between the capsid and the envelope exists an amorphous protein matrix known as the tegument that contains a number of structural proteins, foremost of which is VP16 [4] that acts in concert with cellular transcription factors Oct-1 and HCF to activate HSV immediate–early (IE) gene promoters. Transcription of the IE transcriptional regulatory genes thereby activates the remainder of the lytic life cycle cascade of gene expression that ultimately results in the production of progeny virus particles and the lysis of the infected cell. In addition to VP16, the tegument also contains the UL41 (virion host shutoff, vhs) gene product involved in the shutoff of host protein synthesis, thereby aiding the preferential translation of viral messages [5].

HSV Vector Engineering

a

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HSV Genome VP16

Essential Accessory

UL LAT ICP0

b

UL41

ICP27

ICP4

ICP4

US

LAT ICP22 ICP0

ICP47

HSV Particle Envelope Glycoproteins

Viral dsDNA Genome Viral Capsid Tegument Proteins

Fig. 1 Organization of the HSV-1 genome and structure of the virion particle. (a) Schematic representation of the HSV-1 genome, showing the unique long (UL) and unique short (US) segments, each bounded by inverted repeat elements. The locations of the VP16, ICP27, and ICP4 essential genes that are required for viral replication in vitro are indicated above the viral genome, while the ICP0, LAT, UL41, ICP22, and ICP47 nonessential genes, which may be deleted without dramatically affecting replication in tissue culture, are depicted below the genome. (b) Electron microscopic depiction of the HSV virion showing the icosahedral-shaped nucleocapsid containing the 152 kb double-stranded viral genome; the tegument which contains VP16, UL41, and other HSV-encoded gene products; and the envelope containing the virus-encoded glycoproteins that are responsible for the attachment and entry of the virus into receptor-bearing cells

During natural infection in the human host or in animal models of virus infection, the virus initially replicates in epithelial cells of the skin or the mucosa usually resulting in lysis of these cells. Progeny virions from this initial infection are then taken after attachment and entry of sensory nerve termini of the peripheral nervous system (PNS) and carried via retrograde axonal transport to peripheral nerve cell nuclei where the viral DNA genome is injected through a modified capsid penton portal into the nucleus, after which two alternative forms of the viral life cycle may ensue. The virus may enter the lytic form of the replication cycle, in which expression of viral IE genes serves to transactivate expression of early (E) genes whose products are the principal components of the viral DNA replication machinery that ultimately leads to the production of concatemers of the viral genome. Following viral DNA synthesis, in conjunction with IE gene products, the late (L) genes that encode the structural proteins such as the capsid, tegument, and viral glycoproteins present within the virion envelope are then transcribed. These late genes are required for viral particle assembly within the nucleus, budding of the particle through a modified portion of the nuclear membrane, transport of that particle to the cell surface, and egress from the cells with release of fully infectious progeny virus

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particles. Compared to the process of lytic infection, the virus may enter a latent state, in which the over 85 viral gene products characteristic of lytic infection are either not transcribed or are transcriptionally silenced over time by methods that are not completely understood but are thought to involve genome methylation and histone binding and acetylation. The ability of the virus to enter either the lytic or the latent stage of the virus life cycle holds true for replication-competent (oncolytic) and replication-defective vectors; however replication-defective vectors have been rendered deficient through the deletion of one or more essential HSV gene products, usually IE transcriptional regulatory factors such as infected cell polypeptide (ICP) 4 and 27, making these viruses unable to replicate, and thus they directly enter a “latent-like” state where the viral genome persists for a long term along with the expression of the latency-associated transcripts or LATs [6], the real hallmark of HSV latent infection of the nervous system. HSV possesses numerous biological features that make it attractive as a gene delivery vehicle for gene transfer to the nervous system and other tissues [7–9]. The virus possesses a broad host range and is able to infect both nondividing cells such as neurons and dividing cells at extremely high efficiencies [9–12]. The virus is capable of establishing a latent infection in neurons as part of the natural biology of the virus, a state in which viral genomes persist as intranuclear episomal elements and become transcriptionally silenced over time. Completely replication-defective viruses can be constructed which retain the ability to establish a latent-like state in neurons but which are unable to replicate or reactivate from this latent-like state in contrast to wild-type virus which may be reactivated from latency. These relatively transcriptionally silent persistent genomes still retain the ability to express transgenes for a long term using the HSV latency viral promoter system [13–15]. The large capacity of the viral genome (152 kb), and the fact that many viral genes can be removed as contiguous segments without dramatically affecting virus production, has enabled the incorporation of large [16] or multiple [17] transgenes making it the sole vector capable of expressing multiple gene products or gene libraries. Since HSV genes are expressed in a sequential, interdependent lytic cycle cascade [18], the simple removal of the essential IE gene ICP4 blocks expression of later downstream viral genes in the gene expression cascade [19] resulting in the production of a first-generation replication-defective vector that is incapable of producing virus particles. Since these first-generation vectors are toxic to some cells in culture [20] due to the expression of the remaining IE genes, second- and third-generation vectors deleted for combinations of these multiple IE genes were engineered that displayed reduced cytotoxicity compared to the first-generation vectors [21–23]. A third-generation vector deleted for ICP4, ICP27, and ICP22 (TOZ.1) containing an ICP0 promoter-lacZ expression cassette exhibited reduced


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toxicity in neurons in culture [21]. Another third-generation vector, E1G6 or vHG [24], is also less cytotoxic. We have developed methods to systematically introduce foreign genes into the HSV-1 genome by homologous recombination [25], initially using the TOZ.1 vector backbone. This vector can only be propagated using the ICP4/ICP27-complementing cell line (7b) that has been engineered to avoid overlap of these sequences with the deletions present within the virus in order to eliminate the chance of homologous recombination and rescue of the mutant viruses during propagation in the complementing line [26]. We have readily constructed a series of vectors using this TOZ.1 vector backbone by homologous recombination of a plasmid containing a cassette expressing the gene of interest inserted into the UL41 gene sequence [21, 25]. We have recently employed a similar vector to TOZ.1, designated E1G6 or vHG [24], that also contains the same deletions of ICP4 and ICP27 (Fig. 2a), yet is not deleted for ICP22 like TOZ.1, since we found that elimination of ICP22 resulted in a 1–2 log reduction in virus titer. Instead, vHG possesses deletions within the ICP22 and ICP47 IE gene promoters that results in these genes being expressed as early rather than IE genes. Although vHG lacks the lacZ reporter gene cassette in the UL41 locus, it possesses an HCMV promoter-driven eGFP reporter gene cassette within the ICP4 loci in place of the deleted coding sequences for ICP4 (Fig. 2a). To aid in the rapid identification of recombinants, we have recently introduced an HCMV-mCherry expression cassette into the UL41 locus of vHG (Fig. 2a). Recombination of targeting plasmids such as pSASB3, that contains ICP4 flanking sequences for homologous recombination on either side of a multi-cloning site flanked by promoters (HCMV, HSV LAP2 latency promoter, or the hybrid LAP2-HCMV promoter) and a bovine growth hormone (BGH) polyadenylation sequence (pA) (Fig. 2b), into the ICP4 loci results in the insertion of the therapeutic gene with the corresponding loss of the eGFP expression cassette, enabling the rapid identification of recombinants due to the loss of green fluorescent signal (Fig. 2c). However, we found that it was difficult to identify recombinants that produced clear plaques in the background of green plaque-producing parental virus. So to further aid in the identification of recombinants, we have introduced an HCMV-mCherry expression cassette into the UL41 locus of vHG (Fig. 2a), designated vHG-mCherry, that enables the easy identification of mCherry+/eGFP− plaques in the background of mCherry+/ eGFP+ plaques produced by the parental virus. Moreover, inclusion of two fluorescent reporter cassettes within the virus allows for the recombination of genes into either or even both loci in circumstances that require the introduction of multiple genes into the vector. Following three rounds of limiting dilution analysis, the structure of the recombinants is then confirmed by Southern blot, PCR, or sequence analysis. We have also developed detailed methodologies for the production and purification of large-scale stocks of HSV

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William F. Goins et al. β−ICP47

β−ICP22

ΔICP27

ΔUL41

a vHG-mCherry

ΔICP4

HCMV mCherry IEp

HCMV IEp

SV40 pA

GFP

BGH pA

ΔICP4

BGH pA

GFP

HCMV IEp

b pSASB3 -LAP2 or –HCMV or LAP2-HCMV ICP4 5’ flanking sequence

ICP4 3’ flanking sequence

HindIII—LAP2– BamHI-SpeI-EcoRI-PstI-EcoRV-NotI-XhoI-SphI-XbaI-BGHpA-XbaI -HCMV-LAP2-HCMVΔUL41

c vH-Therapy Gene

βICP22

ΔICP27

ΔICP4

HCMV SV40 mCherry IEp pA

d

HCMV Therapeutic BGH pA Gene IEp

βICP47 ΔICP4

BGH Therapeutic HCMV pA Gene IEp

1) Obtain plasmid clone containing therapeutic gene of interest 2) Subclone into ICP4 (pSASB3) or UL41 (p41) transfer plasmids 3) Verify clones by restriction digestion and/or sequencing 4) Prepare MaxiPrep of new plasmid construct 5) Transfect transfer plasmid into 7b cells twice, then infect with vHG-mCherry virus 6) Wait until CPE occurs, then harvest cells + supernatant 7) Perform limiting dilution analysis of harvest 8) Select mCherry+/GFP− (ICP4) or mCherry−/GFP+ (UL41) plaques 9) Screen isolates for presence of therapeutic gene (Southern, Western, ELISA, IHC)

Fig. 2 Construction and production of a replication-defective recombinant HSV-1 vector. (a) Replicationdefective HSV-1 vector vHG-mCherry, deleted for ICP4 and ICP27, with both ICP22 and ICP47 expressed as early genes (β-ICP22/β-ICP47), contains an HCMV promoter-driven eGFP expression reporter gene cassette in the ICP4 loci and an HCMV-driven mCherry reporter gene cassette within the UL41 locus. This parental virus vector produces both green and red plaques when plated on the 7b complementing cells. (b) The therapeutic gene of interest is cloned into the unique BamHI restriction site within the pSASB3 transfer plasmid downstream of the HCMV, LAP2, or hybrid LAP2-HCMV promoters and upstream of the BGH polyadenylation signal (pA). The pSASB3 plasmid possesses over 1 kb of ICP4 flanking sequences on either side of the BamHI site to ensure homologous recombination into the ICP4 loci of vHG-mCherry. (c) Homologous recombination of the gene of interest within the pSASB3 transfer plasmid into the ICP4 loci of vHG-mCherry will result in a vector that shows a eGFP−/mCherry+ plaque phenotype compared to the eGFP+/mCherry+ plaque phenotype of the parental vHG-mCherry vector. (d) The various steps of the process of inserting your gene of interest into the vHG-mCherry vector by homologous recombination are detailed

vectors [27, 28]. Although the methods detailed in this chapter concentrate on the generation and use of replication-defective HSV vectors, these techniques can also be applied to replication-competent vectors except that they do not require a complementing cell line for their growth and propagation.


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Materials Cell Culture

1. DMEM–10 % FBS: Dulbecco’s Eagle’s modified essential medium (DMEM) supplemented with nonessential amino acids, 100 U/mL penicillin G, 100 μg/mL streptomycin sulfate, 2 mM glutamax, and 10 % fetal bovine serum (FBS). Store at 4 °C. 2. Serum-free DMEM. 3. Methylcellulose overlay (1.0 %): Add 25 g methylcellulose to 100 mL phosphate-buffered saline (PBS) pH 7.5 in a 500 mL sterile bottle containing a stir bar. Autoclave the bottle on liquid cycle for at least 45 min. After the solution cools, add 350 mL of DMEM supplemented with nonessential amino acids, 100 U/mL penicillin G, 100 μg/mL streptomycin sulfate, and 2 mM glutamax; mix well; and place the bottle on a stir plate at 4 °C overnight. Once the methylcellulose has entered solution, add 50 mL of FBS. Store at 4 °C (see Note 1). 4. 1 % crystal violet solution (in 50:50 ethanol:dH2O v/v): Dissolve 1 g crystal violet in 50 mL dH2O, and then add 50 mL of ethanol. Filter using a 0.22 μm filter, and store at room temperature.

Cells

1. Vero (African green monkey kidney; ATCC#CCL81, Rockville, MD) cells or 7b complementing cells that express both ICP4 and ICP27 [26] are required to propagate HSV-1 replicationcompetent or replication-defective viruses.

2.3 Buffers and Solutions

1. Tris-buffered saline (TBS) pH 7.5: 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM ethylenediamine tetraacetic acid (EDTA). Store at room temperature.

2.2

2. PBS (1×) pH 7.5: 135 mM NaCl, 2.5 mM KCl, 1.5 mM KH2PO4, 8.0 mM Na2HPO4 pH 7.5. Store at room temperature. 3. Glycerol. Store at room temperature. 4. 70 % ethanol. Store at −20 °C. 5. Lipofectamine 2000 (Life Technologies). Store at 4 °C. 6. Opti-MEM (Life Technologies). Store at 4 °C. 7. 5 M NaCl. Store at 4 °C. 8. 100 mg/mL dextran sulfate MW9-20 K. Store at 4 °C. 2.4

Nucleic Acids

1. Transfer plasmid pSASB3 (Fig. 2b) for recombination into the ICP4 loci. Other transfer plasmids such as p41 can also be employed [21, 25] that will enable the transfer of the expression cassette into the UL41 locus of the vector. 2. Plasmid containing the gene of interest. 3. E1G6-mCherry (vHG-mCherry) virus (Fig. 2a).

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2.5


Equipment

1. 6,360-cm2 roller 10-layer Cell Stack (Corning, Corning, NY). 2. 6-well, 12-well, and 96-well flat-bottomed plates. 3. T-75 and T-150-cm2 flasks. 4. Cell scrapers. 5. 15- and 50-mL conical polypropylene tubes. 6. Cup-horn sonicator (Virtis, Gardiner, NY). 7. Nutator rocking platform (Clay Adams, Becton-Dickinson). 8. 50- and 500-mL polypropylene centrifuge bottles (Beckman). 9. Multichannel pipetor. 10. Mini-Prep kit (Qiagen, Valencia, CA). 11. Parafilm. 12. 0.8-μm CN vacuum filter for small samples up to 100, 250, 500, or 1,000 mL bottle filters (Nalgene-Thermo/Fisher, Pittsburgh, PA). 13. 1.5-mL cryovials.

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Methods The protocols contained here describe the methods necessary to construct and purify recombinant genomic HSV vectors. Although the chapter details the procedures for constructing and producing a replication-defective HSV vector, these same methods can be applied to replication-competent genomic HSV vectors like the oncolytic vectors employed in the glioblastoma multiforme (GBM) and malignant melanoma clinical trials [2, 29, 30]. We have also provided methods for the production and purification of high-titer vector stocks once an isolate is identified and purified through three rounds of limiting dilution analysis. We focus here on the construction of replication-defective vectors, but the same series of steps could be employed for the engineering of replication-competent genomic HSV vectors. The only major difference between the two is that the replicationdefective vectors require the use of a cell line that expresses HSV gene products that are deleted from the genome of the vector in trans to complement the missing essential genes.

3.1 Construction of Recombinant Virus

In order to engineer the desired recombinant virus, the gene of interest to be inserted into the virus must first be cloned into the transfer plasmid (pSASB3 or p41) that contains at least 500– 1,000 bp of flanking HSV-1 sequences (Fig. 2b). In the example delineated in this chapter, we employ the pSASB3 transfer plasmid that contains HSV flanking sequences that enable recombination of the gene expression cassette into the ICP4 gene loci of the vHG-mCherry vector (Fig. 2a) that will result in the loss of


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the GFP reporter with positive isolates screened for the eGFP−/ mCherry+ phenotype. The addition of 500–1,000 bp of flanking sequence is needed to achieve a higher frequency of producing and isolating the recombinant. Flanking sequences as small as 100–200 bp will produce recombinants, but at a very reduced frequency. The pSASB3 and p41 plasmids each contains a unique BamHI restriction site for cloning of the expression cassette into the transfer plasmid. The expression cassette should consist of the cDNA of interest driven by the promoter of interest as well as a polyadenylation signal. Additionally, one can simply use versions of pSASB3 that possess the HCMV, LAP2, or LAP2-HCMV hybrid promoters and a BGH polyA site (Fig. 2b). As an alternative to using pSASB3, the p41 transfer plasmid can be employed containing HSV-1 flanking sequences for recombination into the UL41 locus of vHG-mCherry with the resulting recombinants being eGFP+/mCherry−, in a manner similar to recombination into the UL41 locus of the TOZ.1 vector that contained a lacZ reporter rather than mCherry in UL41 [21, 25]. Initial studies were performed with vHG, which lacks a second reporter gene cassette within the viral vector, so that recombination of the target plasmid into the ICP4 loci again resulting in the loss of the eGFP reporter leads to the production of a clear plaque phenotype that is difficult to screen for in the background of nonrecombinant vHG plaques that appear bright green under fluorescence. Thus, in order to readily detect the recombinants containing the desired therapeutic gene of interest, the parental virus backbone should possess two fluorescent reporter gene cassettes (eGFP, mCherry) at the desired site of recombination (ICP4, UL41). Positive recombinants obtained from the recombination of the therapeutic gene present within the pSASB3 transfer will produce eGFP−/mCherry+ plaques (Fig. 2c) compared to the eGFP+/mCherry+ plaque phenotype of the parental virus enabling rapid identification (Fig. 2a). 1. Clone your gene of interest into the pSASB3 shuttle plasmid at the BamHI site (see Note 2). 2. One day prior to transfection, seed 5 × 105 7b cells in a 6-well tissue culture plate in DMEM/10 % FBS. This will ensure that cells are nearly confluent the next day. 3. Transfect the cells with the plasmid DNA mix using Lipofectamine-2000 in Opti-MEM, following the manufacturer’s instructions. It is important to linearize the plasmid construct before transfection to increase the recombination frequency compared to that obtained with uncut supercoiled plasmid. Digestion of the plasmid to release the insert, followed by purification of the restriction fragment, does not increase the recombination frequency. Although the frequency is the same, use of purified fragment is superior since no chance exists for the insertion of plasmid vector sequences into the virus by semi-

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homologous recombination with any complementary sequences such as promoters or polyadenylation sites (see Note 3). 4. After incubation steps, add fresh DMEM/10 % FBS and incubate at 37 °C. 5. At 24 h post-transfection, repeat the plasmid transfection process and incubate at 37 °C. 6. At 24 h after the second plasmid transfection step, infect with the vHG-mCherry virus at a multiplicity of infection (MOI) of 1–3 virus PFU per cell in 1 mL serum-free DMEM for 60–90 min at 37 °C. After the infection period, add 4 mL DMEM–5 % FBS and re-incubate at 37 °C. 7. It usually takes 2–5 days for plaques to develop depending on the virus and the cell line. You can usually see some signs of CPE within 24–48 h post-infection due to the presence of the fluorescent reporter gene that enables the identification of virus-infected cells. 8. Screen the plate under the fluorescent microscope looking for the presence of eGFP−/mCherry+ isolates in the background of eGFP+/mCherry+ parental virus plaques (see Note 4). 9. Once plaques have formed, harvest media and cells using a cell scraper and transfer into a 15-mL conical tube. 10. Subject cells/media to three cycles of freeze/thaw, and sonicate the cells three times for 15 s each on setting five using a cup-horn sonicator. 11. Centrifuge at 2,060 × g for 5 min at 4 °C to remove cell debris. 12. Store supernatant at −80 °C for use as a stock (see Note 5). 3.1.1 Determine the Titer of the Stock of Recombinant Virus

1. Prepare a series of tenfold dilutions (10−2 to 10−10) of the virus stock in serum-free DMEM media. 2. Add 100 µL of each dilution to a well of a 12-well tissue culture plate containing 4 × 105 7b cells/well (see Note 6). 3. Incubate the plates at 37 °C in a CO2 incubator for 1 hour, then add 1 mL DMEM/10 % FBS, and place in the incubator overnight. 4. Within the next 24 h, remove the media and overlay the monolayer with 1 mL of 1 % methylcellulose/10 % FBS solution to limit virus spread and produce readily visible plaques. 5. Incubate the plates for 3–5 days until well-defined plaques appear, depending on the virus and the cell line used. The presence of the fluorescent reporter gene within the virus readily accentuates the visualization of infectious centers and plaques. 6. Aspirate the methylcellulose overlay and stain with 1 % crystal violet solution (in 50:50 ethanol:dH2O v/v) for 5 min. Remove stain, rinse with water, and air-dry.


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7. Count plaques, and calculate the number of plaque-forming units per 1 mL of original stock (see Note 7). 3.1.2 Limiting Dilution Analysis to Isolate and Purify Recombinants

1. Add 30 PFU of titered original stock virus to 1 mL containing 2 × 106 7b cells in suspension (DMEM/10 % FBS) in a 15-mL conical tube, and place the tube on a Nutator rocker platform at 37 °C for 1.5 h. Cover the cap with parafilm to prevent leaking and contamination. 2. Add 9 mL of fresh DMEM/10 % FBS media, mix, and plate 100 µL in each well of a 96-well flat-bottomed tissue culture plate using the multichannel pipettor. 3. Incubate the plates at 37 °C in a CO2 incubator for a period of 3–5 days until plaques appear, depending on the virus and cell line employed. Again the presence of the fluorescent reporter facilitates this step. Score the wells for the number of plaques. Theoretically, there should be approximately 30 individual plaque wells/plate. Most wells should lack plaques, while some may have two or more plaques. 4. If recombination between the transgene cassette with the ICP4 (or UL41, depending on the transfer plasmid employed) flanking sequences and the virus has occurred, the gene of interest will have replaced the eGFP (or mCherry) reporter gene. When inserting genes into the ICP4 loci, the corresponding positive recombinants should produce the eGFP−/mCherry+ plaque phenotype, while the parental vHG-mCherry virus will show an eGFP+/mCherry+ plaque phenotype. 5. Wrap the plate with parafilm, and store that plate at −80 °C for use as a stock for the next round of limiting dilution. Alternatively, one can just store the cells and media from wells displaying the eGFP−/mCherry+ plaque phenotype. 6. Score wells that have eGFP−/mCherry+ plaques. 7. Select a well containing only single eGFP−/mCherry+ plaques, as these were formed from virus recombinants in which the gene of interest has replaced eGFP (or mCherry if using the p41 transfer plasmid) in vHG-mCherry. 8. Carry out at least two additional rounds of limiting dilution/ plaque isolation using the stock of virus stored at −80 °C, as in steps 1–5 above. At the final round of limiting dilution, all the plaques identified on the plate should show the desired plaque phenotype (i.e., red but not green plaques for insertion of genes into the ICP4 loci of vHG-mCherry). At this point, the virus stock can be used to produce a midistock for the eventual preparation of a high-titer stock for general experimental use. At the same time this stock can be used to produce viral DNA to confirm the presence of the

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insert as well as the absence of the deleted sequences by Southern blot or PCR analyses. 3.2 Virus Stock Preparation and Purification

The following procedure entails the preparation of a virus stock from one 10-layer cell stack factory 6,360 cm2 worth of cells that can be scaled up or down depending on specific needs. We have employed a salt-release treatment step in our production runs as this increases the overall yield of virus in the supernatant fraction two- to tenfold [27, 28]. Moreover, we have now incorporated the addition of dextran sulfate along with the salt-release step to increase our yield (20–200×) based on the production of an HSV2-based vaccine vector [31]. Our new purification protocol (Fig. 3b) calls for the use of filtration steps that can be employed to separate the virus from cellular debris in combination with a centrifugation step to concentrate the filtrate. The ultimate goal is to purify virus particles away from cellular and extracellular debris which was a problem using our older purification procedure (Fig. 3a). Additional downstream purification steps may be added to further eliminate contaminating cellular DNA and protein such as treatment with Benzonase. 1. Seed one 10-layer cell stack with 1.4 × 108 complementing cells in 1,400 mL DMEM/5 % FBS and incubate at 37 °C in a CO2 incubator. 2. Allow cells to become 80–100 % confluent. If overconfluent, lower overall virus yield will be achieved. 3. Infect cells in a small volume using very low MOIs, usually 0.001–0.005 depending on the cell type and virus. For a tenlayer 7b cells infect with virus in a total volume of 300 mL of serum-free media. Make sure that equal amounts of the inoculum spreads to each layer of the ten-layer cell stack (see Note 8). 4. Infection should proceed at 37 °C for 60–90 min, with rocking of the cell stack every 15 min to ensure that the volume covers the monolayer. 5. After the 60–90-min period, add fresh media back up to the desired volume using the media of choice and desired %FBS. For a ten-layer cell stack of 7b cells we use a final concentration of 2 % FBS in a total volume of 800 mL (see Note 9). 6. Re-incubate the ten-layer cell stack at 37 °C overnight. 7. The following day, switch the ten-layer cell stack to 33 °C (see Note 10). 8. Observe the flask daily for the presence of CPE. If virus contains a fluorescent marker it is easy to follow the infection. 9. Harvest once most cells show CPE (90–100 %), have rounded up, and are no longer adherent to the plastic, depending on cell type.


a

Old Virus Purification Method

b

New Virus Purification Method

Harvest

Dextran Sulfate and 0.45M NaCl Treatment

Low speed centrifugation

Harvest

High speed centrifugation

Low speed centrifugation

Resuspend in PBS OptiPrep gradients

Harvest bands

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0.8 µ filtration High speed centrifugation Resuspend in PBS add glycerol to 10%

Vial in 5-200 µL aliquots, store @ -80°C, titer

Fig. 3 Comparison of the HSV vector production and purification procedures. (a) The previous methods employed to obtain purified HSV vectors employed a series of centrifugation steps cumulating with an Optiprep/iodixanol gradient step. We determined that the integrity of viral membrane was dramatically damaged by multiple centrifugation steps and that the density gradient failed to sufficiently separate the viral particles away from small cellular membrane vesicles resulting in a considerable amount of cellular contaminants within the purified vector preparations. Thus we have developed a new strategy (b) for virus production and purification. This new methodology employs salt and dextran sulfate treatment to achieve greater release of virus particles from cellular membranes of infected cells as well as filtration methods for virus separation from cellular debris

10. Add 5 M NaCl to make the overall concentration 0.45 M, and add dextran sulfate solution to a final concentration of 100 μg/ mL; incubate overnight at 33 °C. 11. The following morning, switch from 33 °C to RT and place onto shaking platform for a minimum of 60–90 min or longer. At this point all cells should have detached from the monolayer. 12. Spin down cells and debris by low-speed centrifugation at 2,060 × g at 4 °C for 5–10 min in a refrigerated tabletop centrifuge in 50-mL conical polypropylene tubes. 13. Remove supernatant, and filter through a 0.8-μm CN vacuum filter (see Note 11). 14. Pellet the virus from the supernatant using a high-speed spin (18,600 × g) for 45 min at 4 °C in a refrigerated preparative centrifuge in 50- or 500-mL polypropylene centrifuge tubes/ bottles. A visible white pellet should be present in each bottle after pelleting the virus.

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15. Wash 1× with 1× PBS to eliminate any residual salt. 16. Resuspend the virus in as small a volume of 1× PBS as possible, and leave the tube/bottle at an angle overnight at 4 °C so that the liquid covers the visible virus pellet (see Note 12). 17. Once the pellet is resuspended make sure that you no longer see chunks or particulates. Next, add glycerol (0.22 μM filtered) to 10 % of the total volume, mix, aliquot into 1.5-mL cryovials, and store at −80 °C. We usually aliquot in at least two different volume sizes, for example 5 or 10 μL and a larger size like 50 or 100 μL (see Note 13). 18. Select at least one cryovial from the −80 °C virus stock to titer according to the virus titration protocol (see Subheading 3.1.1). We also confirm the presence of the therapeutic gene by Southern blot, PCR, or sequencing of the insert in the purified virus stock. In addition, we confirm the expression of the therapeutic gene using Western blot, ELISA, IHC, or other functional assays depending on the therapeutic gene inserted into the vector.

4

Notes 1. Stir methylcellulose overlay media before each use as methylcellulose tends to settle at the bottom of the bottle. 2. The transfer plasmid DNA can be prepared by a variety of methods. Large-scale plasmid preparations are not necessary as plasmid DNA prepared using Mini-Prep kits such as the Qiagen Mini-kit is of sufficient purity to deliver high transduction efficiencies. 3. The choice of specific transfection reagent is crucial, as some transfection reagents (Lipofectamine Plus, Fugene, etc.) work poorly or not at all when transfecting large DNAs such as the 152 kb HSV genome, while Lipofectamine, Lipofectamine 2000, and the standard calcium phosphate method work very efficiently. 4. When examining plates under the fluorescence microscope using the filters for red fluorescence the eGFP−/mCherry+ plaques will only show a brighter red signal than those from the parental eGFP+/mCherry+ virus. 5. It is not necessary to add glycerol up to 10 % to the virus supernatant as the medium contains 5 % FBS and proteins which act as a cryoprotectant. 6. In order to obtain a more accurate titer, the titration should be performed in duplicate or triplicate. 7. In calculating the average PFU/mL of the recombinant virus stock, it is important to determine the average number of plaques counted at a specific dilution and then multiply by the


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dilution factor. Since in this example one plated 100 μL of virus stock at each dilution, the dilution factor for the calculation is 10. So the overall titer in PFU/mL = the average number of plaques × the dilution factor to the power of the dilution wells counted. 8. It is important to employ low MOIs to generate the virus stock, as high MOIs result in the introduction of unwanted mutations throughout the viral genome. 9. It is crucial to keep the total volume as small as possible as this determines the overall amount of fluid that one must process during purification steps. Also, it is equally crucial to use a sufficient volume to ensure coverage of the entire monolayer of cells on each layer of the ten-layer cell stack. 10. It is crucial to switch the infected cells from 37 to 33 °C as we have shown that the virus is more stable at 33 °C versus 37 °C, and cell growth is more limited at 33 °C helping to produce virus of a greater yield on a per cell basis. 11. If one employs 0.45-μm filters, one loses a reasonable percentage of the virus yield and one also gets shearing of virus envelopes. The 0.65-μm size is most ideal, but syringe and filter flasks of the 0.65-μm pore size are not commercially available. One can purchase boxes of individual 0.65-μm filters and place into metal filtration units for filtration. The problem is that these are NOT disposable one-time-use filters. Thus, one has to properly clean and sterilize the metal unit after different virus preparations and demonstrate that no contaminant exists. Remember that if you use media containing serum for the infection, the serum will readily cause the filters to clog, so we use media without serum once we begin the infection process, even for viruses that grow poorly. Otherwise you will go through a considerable number of filters during purification. 12. It is important to thoroughly resuspend the pellet in order to get an even suspension of particles; however, vortexing is not recommended as it can damage the particles and render them noninfectious. 13. If virus does not resuspend in the volume of PBS added, consider adding additional sterile PBS until pellet resuspends completely.

Acknowledgements This work was supported by NIH grant P01 DK044935 (Glorioso)Viral Vector Core B (Goins) and P01 CA163205 (Caliguri/ Chiocca)-Viral Vector Core B (Goins). We also thank Drs. Krisky, Wolfe, Wechuck, Ozuer, and Kopp for their contribution to HSV vector production and purification methodologies.

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