RETROVIRAL AND LENTIVIRAL VECTORS FOR GENE THERAPY

Retrovirus History (Key Points) 1st retrovirus discovered = Rous sarcoma virus (RSV): sarcomas in chickens 1st oncogenes of mammals discovered = mouse mammary tumor virus (MMTV) and the Gross mouse leukemia virus 1970s: first use of gene delivery by a virus by Paul Berg (modified SV40 virus from a lambda bacteriophage to infect monkey kidney cells in vitro) 1st pathogenic human retrovirus discovered = Human T-Cell Leukemia virus (1981) 1983: HIV (Human Immunodeficiency Virus) discovered 

Retrovirus Classification Family: Retroviridae Genus Features Examples 1. Alpharetrovirus Simple, Onco Avian leucosis virus, RSV 2. Betaretrovirus Mouse Mammary Tumor Virus 3. Gammaretrovirus Murine leukemia virus (Moloney, Harvey) 4. Deltaretrovirus Complex, Onco Bovine Leukemia, Human T Cell Leukemia (HTLV) 5. Epsilonretrovirus Walleye Dermal Sarcoma 6. Lentivirus Complex HIV, Visna, EIAV 7. Spumavirus Simian Foamy Virus

Schematic representation of a retrovirus particle structure.

Retroviral genome Based on the genome structure, retroviruses are classified into simple (e.g. MLV, murine leukemia virus) or complex retroviruses (e.g. HIV) Both encode four genes: gag (group specific antigen), pro (protease), pol (polymerase) and env (envelope)

Retroviral genome The gag sequence encodes the three main structural proteins: MA (mactrix), CA(capsid), NC (nucleoproteins). The pro sequence, encodes proteases (PR) responsible for cleaving Gag and Gag-Pol during particles assembly, budding and maturation. The pol sequence encodes the enzymes RT(reverse transcriptase ) and IN (integrase), the former catalyzing the reverse transcription of the viral genome from RNA to DNA during the infection process and the latter responsible for integrating the proviral DNA into the host cell genome. The env sequence encodes for both SU ( surface glycoprotein ) and TM (transmembrane) subunits of the envelope glycoprotein. LTRs (long terminal repeats), contain elements required to drive gene expression, reverse transcription and integration into the host cell Chromosome Packaging signal (ψ) required for specific packaging of the viral RNA into newly forming virions A polypurine tract (PPT) that functions as the site for initiating the positive strand DNA synthesis during reverse transcription

REPLICATION CYCLE OF A RETRIVIRUS

Retrovirus replication cycle Attachment of the virion to a specific cell surface receptor Penetration of the virion core into the cell Reverse transcription within the core structure to copy the genome RNA into DNA Transit of the DNA to the nucleus Integration of the viral DNA into random sites in cellular DNA to form the provirus Synthesis of viral RNA by cellular RNA polymerase II using the integrated provirus as a template Processing of the transcripts to genome and mRNAs Synthesis of virion proteins Assembly and budding of virions Proteolytic processing of capsid proteins

Retroviral Packaging Systems Retroviral transfer plasmid encoding your transgene, sgRNA, or shRNA of interest: This sequence is flanked by long terminal repeats (LTRs) that facilitate host genome integration. Packaging genes (viral Gag-Pol): Gag is a structural precursor protein, and Pol is a polymerase. Envelope gene (may be pseudotyped to alter infectivity):The use of the VSV-G envelope provides the widest tropism, or range of cells, a virus can infect. While both lentiviruses and γ-retroviruses use the same genes for packaging (i.e. Gag, Pol, and Env), the isoforms of these proteins, as well as the viral LTRs are different. As a result, lentiviral and γ-retroviral packaging vectors are not interchangeable. General envelope vectors such as VSV-G, however, may be used across both systems.

Packaging using 293T cells This system provides the greatest flexibility to pseudotype γ-retrovirus using different envelope vectors to modify tropism.

Packaging using helper-free packaging cell lines In this method, the cells to be transfected already contain Gag-Pol and/or Env stably integrated, limiting the number of plasmids that must be transfected. For example, Phoenix, a second generation retrovirus producer cell line developed by Garry Nolan, contains Gag-Pol and either an ecotropic envelope, Phoenix-ECO, (for infection of mouse and rat cells), or an amphotropic envelope, Phoenix-AMPHO (for the infection of mammalian cells.) Using this system, virus is produced in just a few days. Another variant, Phoenix-gp, contains only Gag-Pol and permits additional flexibility in pseudotyping.

Viral Psuedotyping: A Double Edged Sword Tropism: The ability of a virus to infect a particular type of host cell Psuedotyping: Altering the viral envelope protein to alter tropism, thus allowing the virus to infect cells it originally could not Tropism Host Range Viral Envelope Protein Receptor for Viral Envelope Ecotropic Mouse / Rat Gap70 mCAT-1 Amphotropic / Dualtropic Mammals 4070A / 10A1 Ram-1 / GALV Pantropic All Animals VSV-G Phosphotidyl serine Phosphotidyl inositol GM3 ganglioside Special care should be used when working with pantropic or amphotropic viruses which can infect humans!

Advantages Long lasting gene expression Efficiently enters cell

Disadvantages Only infects dividing cells Low yield (hard to produce) Potential insertional mutagenesis

Risks Associated with Retroviruses: Insertional Mutagenesis Viral DNA Gene of Interest Virus Oncogene Random integration of viral genome may disrupt endogenous host genes. Of special concern Is disruption of proto-oncogenes, which can lead to increased cancer risk. Target Cell Host Cell DNA Proto-Oncogene

Structure of HIV Virus (Simple but Fatal) Icosahedral (20 sided), enveloped virus of the lentivirus subfamily of retroviruses. Retroviruses transcribe RNA to DNA. Two viral strands of RNA found in core surrounded by protein outer coat. Outer envelope contains a lipid matrix within which specific viral glycoproteins are imbedded. These knob-like structures responsible for binding to target cell.

HIV The outer shell of the virus is known as the Viral enevlope. Embedded in the viral envelope is a complex protein known as env which consists of an outer protruding cap glycoprotein (gp) 120, and a stem gp14. Within the viral envelope is an HIV protein called p17(matrix), and within this is the viral core or capsid, which is made of another viral protein p24(core antigen).

Life cycle of HIV Attachment/Entry 2. Reverse Transcription and DNA Synthesis 3. Transport to Nucleus 4. Integration 5. Viral Transcription 6. Viral Protein Synthesis 7. Assembly of Virus 8. Release of Virus 9. Maturation

HIV genome organization • Single standed (+) sense RNA genome of about 10 kb 5’ cap, 3’ poly(A) tail Stop codon between gag and pol, suppressed by readthrough or frameshifting Looks like mRNA, but does not serve as mRNA immediately after infection Has a direct repeat (R) and unique (U) regions at both ends All retroviruses encode Gag, Pol and Env • Lentiviral genomes encode a number of additional auxiliary proteins, Tat, Rev, Nef, Vif, Vpr and Vpu

Genome of Simple vs. Complex Retroviruses ALV and MLV are “Simple” retroviruses HTLV, HIV, HFV, and WDSV are “Complex” retroviruses that contain accessory genes Notice gag-pol-env in both simple and complex

Regulatory proteins: Tat • HIV LTR functions as a promoter in a variety of cell types in vitro • It includes an enhancer sequence that binds a number of cell type specific transcription activators

Retroviral genome •Retroviruses contain two copies of the RNA genome held together by multiple regions of base pairing • This RNA complex also includes two molecules of a specific cellular RNA (tRNAlys) that serves as a primer for the initiation of reverse transcription • The primer tRNA is partially unwound and hydrogen bonded near the 5’ end of each RNA genome in a region called the primer binding site

Reverse transcription 1) (-) strand synthesis starts near the 5’ end of the (+) strand RNA genome with a specific host tRNA as a primer and runs out of template after ~100 nt 2) Synthesis proceeds to the 5’ end of the RNA genome through the u5 region ending after the r region, forming the (-) strand strong stop DNA (-ssDNA)

Reverse transcription 2) RNA portion of the RNA-DNA hybrid is digested by the RNase H activity of RT, resulting in a single-stranded DNA product 3) This facilitates hybridization with the r region at the 3’ end of the same or second RNA genome, resulting in the first template exchange for RT

Reverse transcription 4) (-) strand DNA terminates at the primer binding site 5) When (-) strand elongation passes the polypurine tract (ppt) region, the RNA template escapes digestion by RNase H and serves as a primer for (+) strand synthesis by DNA dependent DNA polymerization (DDDP)

Reverse transcription 6) (+) strand synthesis then continues back to the U5 region with the (-) strand DNA as the template and terminates after copying the first 18 nt of the primer tRNA and stops, forming the (+) strand strong stop product (+ssDNA)

Reverse transcription 7) The tRNA is then removed by RNase H activity of RT 8) The exposed PBS anneals to the PBS sequence at the 3’ end of the (-) strand DNA, allowing the second template exchange. Product of the second template exchange is a circular DNA molecule with overlapping 5’ ends.

Reverse transcription 9) DNA synthesis is terminated at the breaks in the template strands at the PBS and PPT ends, producing a linear molecule with long terminal repeats (LTRs).

Mechanism of Tat activation • Downstream of the site of initiation of transcription is the TAR RNA sequence which forms a stem loop that binds the 14 kd viral regulatory protein Tat • In the absence of Tat, viral transcription terminates prematurely • Tat facilitates efficient elongation • Tat protein is cytotoxic to cells in culture • Causes depolarization and degeneration of cell membranes • Transgenic expression in mice causes a disease that resembles Kaposi’s sarcoma

Stimulation of transcription by HIV-1 Tat protein: • Before Tat is made proviral transcripts are terminated within 60 bp of the initiation site • Production of the Tat protein allows transcription complexes to synthesize full length RNA • Binding of Tat to TAR together with the cyclin T subunit of Tak leads to stimulation of phosphorylation of the largest subunit of RNA polymerase II • As a result, the transcriptional complexes become competent to carry out transcription

Regulatory proteins: Rev • Rev Protein is an RNA binding protein that recognizes a specific sequence within the structural element in env called the Rev-responsive element (RRE)

Rev protein: • Rev activates the nuclear export of any RRE containing RNA • As the Rev concentration increases, unspliced or singly spliced transcripts containing the RRE are exported from the nucleus • Rev facilitates synthesis of the viral structural proteins and enzymes and ensures availability of full length genomic RNA to be incorporated into new virus particles • The accessory proteins, Vif, Vpr and Vpu are also expressed later in infection from singly spliced mRNAs and their export to the cytoplasm is Rev dependent

HIV accessory proteins Nef protein: • Translated from multiply spliced early transcripts • myristylated at its N-terminus and anchored to the inner surface of the plasma membrane • Nef deleted HIV and SIV are much less pathogenic in vivo • Nef downregulates expression of CD4 by enhancing endocytosis • Can activate CD4+ T lymphocytes by modulating signaling pathways

HIV accessory proteins Vif Protein: • Viral infectivity factor • Accumulates in the cytoplasm and at the plasma membrane of infected cells • Mutant viruses lacking the vif gene were less infectious and defective in some way • vif-defective virions enter cells, initiate reverse transcription, but do not produce full-length double stranded DNA • vif inhibits antiviral action of a cytidine deaminase, which is synthesized in nonpermissive cells This enzyme deaminates deoxycytidine to deoxyuridine and leads to endonucleolytic digestion or G to A transitions

1st Generation Lentivirus Vectors Transient transfection of three plasmids in 293T : Packaging plasmid: all HIV viral genes, except env Envelope plasmid: G envelope glycoprotein of vesicular stomatitis virus (VSV G) Transducing vector: gene or cDNA of interest and the minimal cis-acting elements of HIV

1st Generation Vectors Limited homology between vector and helper sequences Separation of helper plasmids Still retains HIV accessory genes in the packaging plasmid

Packaging Recombinant Lentiviral Particles The three plasmids containing the viral genome components are transfected into the packaging line to create the infectious viral particles. Multiple plasmids are used so multiple recombination events would be required to reconstitute a replication competent virus.

Lentiviral Components To increase the safety of lentivirus, the components necessary for virus production are split across multiple plasmids (3 for 2nd-generation systems, 4 for 3rd-generation systems). The components of both systems are as follows: Lentiviral transfer plasmid encoding your insert of interest. This sequence is flanked by long terminal repeats (LTRs) that facilitate host genome integration. To improve safety, transfer vectors are all replication incompetent and may additionally contain a deletion in the 3'LTR, rendering the virus “self-inactivating” (SIN) after integration. Packaging plasmid(s) Envelope plasmid

2nd Generation This system contains a single packaging plasmid encoding the Gag, Pol, Rev, and Tat genes. The transfer plasmid contains the viral LTRs and psi packaging signal (not pictured). Unless an internal promoter is provided, gene expression is driven by the 5'LTR, which is a weak promoter and requires the presence of Tat to activate expression. The envelope protein Env (usually VSV‐G due to its wide infectivity) is encoded on a third, separate, envelope plasmid. All 2nd generation lentiviral transfer plasmids must be used with a 2nd generation packaging system because transgene expression from the LTR is Tat-dependent.

2nd Generation Vectors Elimination of accessory genes from packaging plasmid No effect on vector titer Retains property of transduction of many dividing and non-dividing cells Increased safety margin

3rd Generation Self-inactivating (SIN) vectors The packaging system is split into two plasmids: one encoding Rev and one encoding Gag and Pol. Although safer, this system can be more cumbersome to use and result in lower viral titers due to the addition of one additional plasmid. Tat is eliminated from the 3rd generation system through the addition of a chimeric 5' LTR fused to a heterologous promoter on the transfer plasmid. Expression of the transgene from this promoter is no longer dependent on Tat transactivation. The 3rd generation transfer plasmid can be packaged by either a 2nd generation or 3rd generation packaging system.

Process of producing infectious transgenic lentivirus 3-4 plasmids are transfected into A293T cells: one transfer vector one or two packaging vector(s) one envelope vector After media change and a brief incubation period, supernatant containing the virus is removed and stored or centrifuged to concentrate virus. Crude or concentrated virus can then be used to transduce the cells of interest.

Virus Production

Advantages Long lasting gene expression Will infect dividing and non-dividing cells

Disadvantages Low yield Potential insertional mutagenesis

What is the difference between a lentivirus and a retrovirus? Lentiviruses are a subtype of retrovirus. From an experimental standpoint the main difference between lentiviruses and standard retroviruses (γ-retroviruses) is that lentiviruses are capable of infecting non-dividing and actively dividing cell types whereas standard retroviruses can only infect mitotically active cell types. This means that lentiviruses can infect a greater variety of cell types than retroviruses. Both lentiviruses and standard retroviruses use the gag, pol, and env genes for packaging; however, they are different viruses and thus use slightly different isoforms of these packaging components. Therefore, lentiviral vectors may not be efficiently packaged by retroviral packaging systems, and vice versa

VSV-G Pseudotyped Lentiviral Vectors Efficiently Transduce Many Cell Types R GFP BGH PA HeLa MCF10A HEK 293 GTM3

VSV-G Pseudotyped Lentiviral Vectors Can Transduce Primary Rat Hepatocytes GFP BGH PA MOI: 100 50 25 10

What safety concerns surround the use of lentiviral vectors? The two main safety concerns surrounding the use of lentiviral are: The potential for generation of replication-competent lentivirus The potential for oncogenesis

What safety concerns surround the use of lentiviral vectors? The potential for generation of replication-competent lentivirus is addressed by the design of the vectors and by safe laboratory practice. 2nd and 3rd generation lentiviral systems separate transfer, envelope, and packaging components of the virus onto different vectors. The transfer vector encodes the gene of interest and contains the sequences that will incorporate into the host cell genome, but cannot produce functional viral particles without the genes encoded in the envelope and packaging vectors. Unless recombination occurs between the packaging, envelope, and transfer vectors, and the resulting construct is packaged into a viral particle, it is not possible for viruses normally produced from these systems to replicate and produce more virus after the initial infection.

What safety concerns surround the use of lentiviral vectors? 3rd generation systems are considered safer than 2nd generation systems because the packaging vector has been divided into two separate plasmids (resulting in a four plasmid system in total). In addition, 3rd generation systems do not use the HIV protein tat in order to produce full length virus from the transfer vector during the viral production stage. Self-inactivating (SIN) vectors vectors have a deletion in the 3'LTR of the viral genome that is transferred into the 5'LTR after one round of reverse transcription. This deletion abolishes transcription of the full-length virus after it has incorporated into a host cell. The potential for oncogenesis is largely based on the specific insert contained within the lentiviral transfer vector (dependent upon whether or not it is an oncogene) and should be considered on a case by case basis. Biosafety should always be considered with respect to the precise nature of experiments being performed

Viral Vector Biosafety Resources The ability of viruses to target and invade cells makes them a key tool for biological research, but also raises biosafety concerns. Chief concerns are 1) the potential generation of replication competent virus and 2) the potential for oncogenesis through insertional mutagenesis. These risks are dependent on the vector system used and the transgene insert encoded by the vector. While the viral vector systems in current use are designed to minimize risk to researchers, it’s important to consider both of these concerns when designing your experiments.

General Biosafety Guidelines Lentivirus: lentiviral systems are derived from HIV, but their organization across multiple plasmids and the deletion of many HIV proteins lowers the probability of generating replication-capable virus. These systems are handled at BSL-2. Retrovirus: Retroviruses are classified based on the cell types they infect. For retroviruses that do not infect human cells, BSL-1 may be appropriate; if human cells can be infected, BSL-2 is appropriate.