In recent years, retroviral and lentiviral vectors have become increasingly vital tools for the delivery of nucleic acids to many cell types in a variety of experimental systems. In addition to being important in laboratory settings for use in both in tissue culture and animal models, they have also been applied in clinical trials to treat genetic diseases. LENMELDY from Orchard Therapeutics has been approved by FDA to treat metachromatic leukodystrophy. It contains autologous CD34+ cells ( hematopoietic stem cells) transduced with a lentiviral vector encoding the human arylsulfatase A (ARSA) gene. Zynteglo from Bluebird Bio is a gene-therapy treatment for transfusion-dependent beta-thalassemia, in which autologous hematopoietic stem cells are transduced ex vivo with the beta-globin gene via a lentiviral vector pseudotyped with vesicular stomatitis virus glycoprotein G. Using lentiviral or retroviral systems allows for the stable, heritable integration of a specific nucleic acid sequence into the target cell’s genome. Utilizing this feature of lentiviral and retroviral vectors allows for the theoretically permanent expression of a gene construct, such as an siRNA or protein coding sequence, in a population of cells, or on a single-cell level [6].

Members of the Retroviridae family, which include γ-retroviruses and lentiviruses, are characterized by their ability to retrotranscribe their RNA genome into a cDNA copy, which is then stably integrated into the host cell genome. Retroviruses are often described as either simple (sometimes called oncogenic or γ-retroviruses; e.g., the murine leukemia virus [MLV]) or complex (e.g., lentiviruses). The main difference between these subtypes is the presence of a number of accessory and regulatory genes in complex, but not simple, retroviruses (discussed further below) [4, 7]. The viral particles of both groups (Figure 1) contain two copies of positive-stranded RNA with an associated viral reverse transcriptase (RT) located within an internal core. Also located within this compartment are structural and enzymatic proteins, including the nucleocapsid (NC), capsid (CA), integrase (IN), and protease (PR). The inner core is surrounded by an outer protein layer comprised of the matrix (MA) protein, which is in turn encompassed by the envelope glycoprotein (ENV)-studded, host cell membrane-derived envelope [4, 7]. Intasome, a tetramer of integrase (IN) assembled on viral DNA ends, captures the host's nucleosomes and enables viral DNA recombination at specific hotspots [8].

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Figure 1. Simple and Complex Retrovirus Virion Structure. The viral particle contains two copies of reverse transcriptase (RT)-associated positive-stranded RNA within the internal core. Also located here are the nucleocapsid (NC), capsid (CA), integrase (IN), and protease (PR). The inner core is surrounded by an outer Matrix (MA) layer which is in turn encompassed by the glycoprotein (ENV)-studded, host cell membrane-derived envelope. adapted from [1-4].

Simple and complex retroviruses both contain two copies of linear, nonsegmented, single-stranded RNA of 7-12 kb in length encoding the gag, pol, and env genes. Gag encodes a polyprotein that is translated from an unspliced mRNA which is then cleaved by the viral protease (PR) into the MA, CA, and NC proteins. The Env gene also encodes a polyprotein precursor which is cleaved by a cellular protease into the surface (SU) envelope glycoprotein gp120 and the transmembrane (TM) glycoprotein gp41. While gp120 interacts with the cellular receptor and coreceptor, gp41 anchors the gp120/gp41 complex in the viral membrane and catalyzes membrane fusion with the host cell during entry. Pol is expressed as a Gag-Pol polyprotein as a result of ribosomal frameshifting during Gag mRNA translation, and encodes the enzymatic proteins RT, PR, and IN. These three proteins are associated with the viral genome within the virion. The RT protein possesses three distinct activities: (a) RNA-dependent DNA polymerase activity, responsible for transcribing the two RNA genomes into a single cDNA; (b) RNase H activity; and (c) DNA-dependent DNA polymerase activity. PR cleaves the gag and Gag-Pol polyproteins, resulting in virion maturation and the production of fully infectious virions. IN is responsible for the integration of viral cDNA into the host cell genome. Once integrated, the viral genome is contiguous with the host cell chromosome and is referred to as a provirus [1, 4, 7, 9].

The main feature of complex retroviral genomes distinguishing them from those of simple retroviruses is the presence of a set of accessory genes whose products are involved in the regulation of transcription, RNA transport, gene expression, and assembly. These include the Rev and Tat proteins as well as the accessory proteins Vpu, Vif, Vpr, and Nef. Rev is an RNA-binding protein that promotes late phase gene expression. It is also important for the transport of the unspliced or singly-spliced mRNAs, which encode viral structural proteins, from the nucleus to the cytoplasm. Tat is an RNA-binding protein that enhances transcription. The Nef protein inhibits T-cell activation. Vpu enhances the release of the virus from the cell surface to the cytoplasm during entry [7]. The Vif protein is necessary for replication of lentiviruses due to its ability to downregulate the host’s antiviral response [7, 10].

The integrated provirus genome (Figure 2) has 5’ and 3’ long terminal repeats (LTR) which each consist of three regions: (a) the U3 region, which functions as a promoter and contains transcriptional enhancer elements and a TATA box; (b) the R region, which is where transcription begins; and (c) the U5 region, which is involved in reverse transcription and carries a tRNA primer-binding site. Other important sequence elements of the provirus are the packaging signal (ψ, psi) and the polypurine tract (ppt), which serves as the site of initiation of positive-strand DNA synthesis during reverse transcription [4, 7, 9].

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Figure 2. Complex Retrovirus Genome Structure. The linear, nonsegmented, single-stranded RNA of retroviruses is 7-12 kb in length and encode the genes gag, pol, and env. The gag gene encodes a polyprotein that is translated from an unspliced mRNA which is then cleaved by the viral protease (PR) into the MA, CA, and NC proteins. The env gene also encodes a polyprotein precursor which is cleaved into the surface (SU) envelope glycoprotein gp120 and the transmembrane (TM) glycoprotein gp41. The pol gene is expressed as a Gag-Pol polyprotein and encodes the enzymatic proteins RT, PR, and IN. These three proteins are associated with the viral genome within the virion. Unlike simple retroviruses, complex retroviruses encode a series of accessory proteins including Vpu, Vif, Vpr, and Nef. The integrated provirus genome has 5’ and 3’ long terminal repeats (LTR) which consist of U3, R, and U5 regions. Finally, the psi (ψ) sequence serves as the packaging signal for the RNA genome. adapted from [1, 2].

Despite the differences in the genome, the replication cycles of simple and complex retroviruses are very similar (Figure 3). Infection begins when the envelope glycoprotein gp120 binds the cellular receptor, resulting in a conformational change in ENV that exposes the TM subunit, resulting in fusion of the virion envelope and cellular membrane and subsequent release of the core into the cytoplasm [4]. Viral tropism is determined by recognition of specific cellular receptors by the viral envelope glycoprotein gp120 [9]. For example, HIV and SIV recognize CD4 on helper T-lymphocytes and macrophages followed by interaction with the coreceptor, most often CXCR4 or CCR5 [1, 9]. While still in the viral core, the RNA genomes are reverse transcribed by RT into double-stranded DNA [1, 2, 11].

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Figure 3. Retroviral Replication. Infection begins when the viral Env protein interacts with the cellular receptor and enters the cell. The RNA genome is reverse-transcribed by RT into dsDNA which then enters the nucleus and is integrated into the host genome through the activity of IN. After the accumulation of newly synthesized viral proteins and viral genomic RNA, the components are packaged and bud from the cell, acquiring a cellularly-derived membrane. The particle matures when the Gag and Gag-Pol polyproteins are cleaved by the viral protease. Figures adapted from [1, 2, 4, 5].

It should be noted that this is the point at which the viral replication cycle of lentiviruses differs from that of simple retroviruses. The viral dsDNA of simple retroviruses is not capable of passing through the nuclear pore complex and requires the breakdown of the nuclear membrane during mitosis for integration of the cDNA to occur [4, 7]. During lentivirus infection, on the other hand, the preintegration complex is transported into the nucleus, and therefore lentiviruses are capable of integration in both dividing and nondividing cells [4, 12]. This is an important feature to consider when selecting the appropriate vector for a specific application, as retroviral vectors will not efficiently integrate the transgene in nondividing cells.

Integration is mediated by the enzymatic activity of IN. The process begins with the removal of several nucleotides from the 3’ termini of both strands of cDNA. IN also cleaves the cellular DNA, and the viral cDNA is ligated to these overhangs. Single-stranded gaps are filled in by the cellular DNA repair machinery [1]. The integrated viral DNA is referred to as the “provirus” and is replicated and inherited along with the host chromosome. For a long time it was thought that integration did not occur in a sequence-specific manner [9]. However, an early indication that integration may occur non-randomly was stumbled upon during a gene therapy trial for X-SCID in which a therapeutic MLV-based vector selectively integrated in close proximity to a proto-oncogene in multiple subjects. This appeared to be a contributing factor to the development of leukemia in some of the recipient patients, and raised concerns about the mutagenic side effects of retroviral insertion. Since the sequencing of the entire genome, recent studies suggest that there are, in fact, virus-specific integration patterns that may be dependent on chromatin structure rather than DNA sequence. For example, it was recently demonstrated that the integration of HIV-1 cDNA is hindered when the target chromatin is compacted [13]. Avian sarcoma virus (ASV), on the other hand, has actually been observed to integrate more efficiently after condensation of the target chromatin. Also, for HIV-1 and -2, HIV-1-based vectors, simian immunodeficiency virus (SIV)-based vectors, and feline immunodeficiency virus (FIV), about 70 % of integration events take place within genes [13]. Lentiviral vectors seem to show a preference for integration at sites distant from transcriptional start sites [11, 14]. In contrast, about 20 % of integration events during MLV (a γ-retrovirus) infection takes place near the 5‘ end of transcription units, with some preference for CpG islands and proximity to DNase I-hypersensitive sites. Interestingly, HIV-1, SIV, MLV and ASLV seem to have symmetric base preferences surrounding the sites of integration [13, 15]. Further complicating our understanding of proviral integration site selectivity is the complex contribution of host cell factors, which varies depending on the retrovirus in question and cell type infected [13]. The biases in site integration can have interesting implications for the level of genotoxicity of various lentiviral or γ-retroviral vectors. Notably, lentiviral vectors appear to have low oncogenic potential when compared to retroviral vectors [11, 14].

The integrated viral cDNA is transcribed by the cellular transcriptional machinery [4] using the viral promoter/enhancer in the 5’ LTR [16]. The transcripts are transported from the nucleus and translated akin to cellular mRNAs [16]. The early transcripts encode Rev, Nef, and Tat, with Tat and Nef each containing introns that are removed in the mature mRNAs [9]. Tat binds to the Transactivation Response Element (TAR) site located in the 5’ LTR stimulating the transcription of longer transcripts [14]. Rev binds to the introns of newly transcribed viral mRNAs. The accumulation of Tat and Rev results in a shift to late transcription, during which mRNAs that include one or both introns are translated. These longer transcripts encode the structural proteins [9].

In the cytoplasm, two viral genomic RNAs associate with replication enzymes and the core proteins assemble around them, forming the virus capsid. As it migrates towards the cell surface, the Gag and Gag-Pol polyproteins are cleaved by the viral protease, resulting in a mature, infectious particle [9, 14]. The mature particle then buds from the cellular membrane, thus acquiring its envelope [7].

Recently, several labs have produced lentiviruses that do not integrate by the traditional retrovirus mechanism, or alternatively do not integrate at all. The latter, called integration-defective lentiviral vectors, contain a mutated IN which lacks enzymatic activity. These lentiviral vectors produce double-stranded episomal DNA circles in the host cell nucleus. Integration-defective lentiviral vectors were developed partially due to concerns surrounding the random integration and insertional oncogenesis (described above) [15]. There also exist lentiviral vector-transposon hybrids which produce stably integrated transgenes by transposition. Finally, there are lentiviral vectors that insert genes by homologous recombination. This has the advantage of decreasing the chance of insertional mutagenesis and allowing for the natural expression pattern of the ‘replacement’ sequence to be maintained [15].

Another major step forward in lentiviral and retroviral vector technology has been their use in animals and humans. Primary cells can be genetically modified using lentiviral or retroviral vectors (e.g., the introduction of GFP-transduced cancer cells or hepatocytes) and then introduced into mice. The cells can then be tracked using various imaging techniques [14]. Lentiviral vectors could also replace the injection of plasmid DNA into fertilized oocytes as a way to produce transgenic animals due to their gene transfer efficiency, the robustness of transgene expression, and increased survival rate in many different animal species [14, 17, 18]. Lentiviral vectors have also shown promise in medical applications. However safety and genotoxicity concerns are a major hurdle to the widespread therapeutic use of viral vectors. There are also manufacturing challenges, such as ensuring the purity and high yield required for clinical grade lentiviral vectors [19]. There have been several deaths as a result of gene therapy protocols which used adenoviral vectors, γ-retroviral vectors, or adeno-associated virus vectors. As mentioned above, despite a successful trial using retroviral vectors to correct X-SCID, many of the patients eventually died from leukemia that was caused by insertional mutagenesis of the transgene [19]. Progress continues to be made in rendering these vectors safer and more useful for clinical trials. For example, successful correction of sickle cell disease in an animal model was recently achieved by transducing hematopoietic stem cells with a lentiviral vector [20]. Interestingly, promising research has also recently demonstrated that HIV-derived vectors for gene therapy might be useful in combating HIV infection itself [21, 22].

Development of novel nucleic acid delivery methods has opened new avenues for therapeutic applications, such as gene replacements. In particular, a new method using the lentiviral vector G1XCGD for expression of the gp91phoxtransgene in myeloid cells has been successfully tested in a mice model [23]. CD34+ cells transduced with the lentiviral vector have been engrafted into the mice and induced therapeutically notable levels of NADPH activity in myeloid cells.

Also, patients with cystic fibrosis (CF) may sufficiently benefit from the therapeutic approach based on lentiviral vectors, which integrate into the genome and produce stable gene expression. A study performed in CF pigs, an experimental animal CF model, has achieved the improvement of CF transmembrane conductance regulator protein (CFTR) functions using a feline immunodeficiency virus-based lentiviral vector [24]. Thus, a restoration of the anion channel disorder was achieved by lentivirus-delivered CFTR. In addition, internal ribosomal entry sites-based lentiviral vectors, which expressed angiogenic and cardioprotective factors and contractile stimulator SERCA2a, have shown significant improvement of cardiac functions when administered into mice with experimentally induced myocardial infarction [25].

Several clinical trials have already shown initial efficacy for therapeutic lentivirus-based approaches. In particular, a significant improvement of the symptoms of Parkinson’s disease has recently been reported by a trial of ProSavin, a lentiviral vector that delivers dopamine [26]. ProSavin has been shown to be well tolerated in patients with Parkinson's disease.

Care should be taken when handling lentivirus and γ-retrovirus-based vectors as they generally originate from human pathogens. The major concerns when working with these vectors are (a) the generation of replication-competent, contaminating lentivirus; (b) the potential for oncogenesis; and (c) the potential toxicity of the transgene. The second and third generation lentiviral systems, which make up most commercially available lentiviral systems, have many safety features that mitigate these risks [27]. In these systems, the packaging and enzymatic proteins are expressed from separate vectors, reducing homology between the packaging constructs and thus reducing the chance of homologous recombination. Furthermore, many lentiviral transfer vectors are self-inactivating due to a deletion in the 3’LTR [28].

Some steps to take when handling viral vectors include:

• Wearing appropriate personal protective equipment (i.e., a lab coat and gloves; some companies suggest double-gloving)
• Working in a Class II laminar flow hood
• Decontaminating all work surfaces (especially after a spill, splash, or aerosol production)
• Application of BL2 or enhanced BL2 containment

Further information about handling lentiviruses and retroviruses, and a description of laboratory biosafety level criteria, is provided by the NIH and the Centers for Disease Control Office of Health and Safety: [29, 30].

Lentiviral and retroviral gene delivery systems exploit aspects of retrovirus replication to provide stable integration of the desired nucleic acid sequence. Whereas transfection of nucleic acids results only in transient transgene expression, the activity of the viral integrase in retroviral and lentiviral-based systems allows for stable integration of the transgene which is then inherited and continuously expressed over repeated cell divisions. A key feature of both lentiviral and retroviral vectors is that they produce replication-defective, or self-inactivating, particles. This allows for delivery of the desired sequence, without continued viral replication in the target cells. More importantly, since some of these are developed from a human virus, it eliminates the dangers associated with the use of a live pathogen. The production of replication defective virus is accomplished through trans-complementation (Figure 4) in which the packaging cells are cotransfected with three separate plasmids (Figure 5) (see below) that together express all of the viral proteins necessary to generate infectious particles, as well as the nucleic acid sequence of interest that will be packaged within them for delivery. While many lentiviral vector systems are based on transduction of two helper plasmids (second generation) with the transfer plasmid, some newer systems (third generation) have the packaging and envelope constructs on three plasmids which are combined with the transfer plasmid. The typical transfer and two helper plasmids used in replication-defective retroviral or lentiviral production are:

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Figure 4. Making Lentiviral and Retroviral Vectors. Lentiviral vectors (left) are created by cotransfection of a packaging cell line (293T/293FT) with the cDNA/shRNA expressing transfer plasmid along with two helper/packaging plasmids which encode the structural and envelope (typically VSV-G) proteins. The packaging cells produce infectious particles, whose genome only encodes sequences from the transfer plasmid, which can be used to transduce the target cells. Retroviral vector production (right) occurs in a similar manner to lentivirus production, with the main difference being that the initial step is transfection with a single plasmid. The transfer plasmid alone is transfected into a packaging cell line (Phoenix) that already contains the helper constructs. adapted from AddGene.

This vector is used to transfer genes of interest into the target cells. There is a deletion of the U3 region and other transcriptionally active sequences from the 3’ LTR, which results in it being a self-inactivating LTR (which is thought to be safer than having an intact/active LTR which could activate genes adjacent to insertion). The 5’ LTR drives expression of the packaged genomic RNA, and the transgene is driven from a promoter within the vector [14].

Gag and Pol: These viral proteins are needed for maturation of the virion. Tat and Rev upregulate transcriptional activity and nuclear export of genomic RNA. The accessory genes have been deleted to increase safety by reducing the probability of recombination (see Safety Section below) [14]. Tat has been removed in newer generations of lentiviral systems and Rev has been placed on a separate vector to increase safety.

Viral vectors can be pseudotyped with coat proteins from other pathogens to alter their tropism. The most commonly used is a fusogenic envelope G glycoprotein of the vesicular stomatitis virus (VSV-G) [31], for example, AddGene pCMV-VSV-G plasmid ( 8454) [32], along with the packing AddGene psPAX2 plasmid ( 12260) [33-36]. Speaking of vesicular stomatitis virus, there are occassions where a vesicular stomatitis virus pseudotype system is preferred over a lentiviral one,for example, for examining the coronaviral entry into host cells [37]. Other common envelope proteins are derived from rabies virus, MLV, Ebola, baculovirus, measles virus, and filovirus. While baculovirus GP64, and Hepatitis E1 and E2 pseudotyping enhances hepatic transduction, filovirus envelope pseudotyping enhances transduction of airway epithelial or endothelial cells. Interestingly, pseudotyping may also affect trafficking of the lentivirus with the rabies virus glycoproteins causing retrograde axonal transport [15].

Note: Due to isoform differences between γ-retroviral and lentiviral gag, pol, and env genes, the packaging plasmids are not interchangeable with the transfer plasmids [38].

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Figure 5. Lentiviral Plasmids. Most lentiviral systems use a transfer plasmid and two helper plasmids. Their features are described in detail in the text. Newer systems may employ three helper plasmids with the rev gene being encoded on a separate plasmid. This is believed to improve safety by further preventing recombination events from producing replication-competent virus. adapted from addgene.

Procedurally, a key difference between lentiviral and retroviral vectors is that with lentiviral vectors, typically the packaging, envelope, and transfer vectors are cotransfected into the packaging cell line, while with retroviral vectors only the transfer vector is transfected into a cell line that already stably carries the other two vectors. For retroviruses, the transfer vector is transfected into Phoenix cells, a cell line based on Human Embryonic Kidney 293T that carries the two helper constructs (env and gag-pol) as episomes. These cells need only be transfected with the transfer vector in order to produce retrovirus (Figure 4, right). Phoenix cells need to be doubly selected every three months with Hygromycin B and Diphtheria toxin for one week to ensure the presence of both helper constructs in all cells. Phoenix-Eco and Phoenix-Ampho are available through ATCC [39, 40], and Invitrogen. Commonly used lentiviral envelope expressing plasmids include Addgene pMD2.G ( 12259), for example [36, 41].

Typically, Human Embryonic Kidney 293T or 293FT cells serve as the packaging line for lentiviruses. Phoenix-ECO has also been used [42]. 293FT cells were designed to produce high titers of lentivirus. Creation of packaging cell lines for lentiviral vectors has proven to be more difficult as the toxicity of the protease (encoded by the pol gene) and VSV-G has been difficult to overcome [15]. Several retrovirus lentivirus production systems have been developed and are available commercially (listed below in the reagents section).

When choosing a transfer plasmid, it is important to take note of the promoter which drives the gene of interest. RNA Pol II promoters (such as CMV) drive protein-coding RNA expression. RNA Pol III promoters (such as H1 or U6), drive expression of shorter transcripts, such as shRNAs.

• The first step in lentiviral vector production is co-transfection of the packaging cells with the transfer, envelope, and gag-pol plasmids (or single transfection of the transfer plasmid in the case of retroviral vector production).
  Note: Packaging cells should not be allowed to reach confluence as this reduces their transfection efficiency. For example, transfection occured at 70% confluency [43].
  Plasmid DNA quality can also affect transfection efficiency. The use of commercial kits, such as the QIAGEN Endotoxin-free Plasmid Kit, can help if plasmid quality is a problem.
• HEK 293T-derived cells are highly transfectable by either calcium phosphate-mediated or lipid-based transfection (see Reagents section below). Depending on the transfection protocol used, the cells may need to be washed, followed by the addition of fresh media within 12 to18 hours. At about 24 hours post-transfection, the media should be removed and replaced with the media to be applied to the target cells. The packaging cells are now allowed to produce virus for the next 48-72 hours. Since lentiviruses are more stable at 32 °C than 37 °C, the packaging cells can be incubated at this temperature until the virus is collected.

• The highest concentration of virus is typically produced between 48-72 hours. To collect the virus, remove the supernatant from the packaging cells and place it in a 15 mL tube. To remove cellular debris, the supernatant can either be centrifuged at 1500 rpm for 5 minutes or it can be filtered through a 0.45 um filter and concentrated via, for example, Clontech Retro-X concentrator [44].
  Note: The purified virus can be stored at -80 °C until needed. However, the titer drops by about 50% with each freeze-thaw cycle.
• Media can be added back on to the packaging cells and the harvest can be repeated out to 96 hours post-transfection if more virus is desired.
  Note: If desired, the titer of the virus can be determined by various methods, as discussed later.

• The virus-containing medium can now be added to the subconfluent target cells. Actively dividing cells will take up the viruses more efficiently; in the case of retroviral vectors, the cells must be dividing or the construct will not reach the nucleus. Depending on your experiment, you may choose a specific multiplicity of infection (MOI) or you can test a range of volumes of the purified virus to determine which gives you the appropriate results.
• Lentiviral and Retroviral transduction can be enhanced by the addition of polybrene [45, 46] (Santa Cruz sc-134220; MilliporeSigma TR-1003-G [47] ; MilliporeSigma 107689 [48] ) or protamine sulphate [41, 49]. Also known as hexadimethrine bromide, this cationic polymer is used to increase the efficiency of retrovirus transduction. It is thought to act by neutralizing the virus-cell charged repulsion [50] and enhancing receptor-independent adsorption of the viruses [51]. For suspension cells (as compared to adherent cells), spinoculation can facilitate the transduction process [44, 48], although it may decrease the survival of, for example, sensitive T cells [52]. Jung HY et al transduced freshly isolated primary mouse mammary epithelial organoids and Caco2 cells/organoids with shRNA lentiviruses and gene-overexpressing retroviruses by using protamine sulphate [49].
• Inclusion of Rho kinase inhibitor Y-27632 can enhance the survival of cells and organoids derived from stem cells during viral transduction [44, 53].
• Change the media the following day
  Note: The media can be changed in a little as 4 hours if the toxicity of the lentiviral particles is a concern.
  Note: Reverse transcription and integration of the construct take place within 24-36 hours.
• The transduction process can be repeated if needed. When the virus-containing medium is removed from the packaging cells, fresh media is replaced. This can be collected 24 hours later and used to repeat the transduction step.

• Some transfer vectors also contain a marker such as a fluorescent reporter or drug selection marker.
• FACS sorting can be used to enrich for cells which express a fluorescent reporter (such as GFP).
• If it contains a drug-selectable marker, follow the protocol for the particular drug. For example, puromycin selection is usually carried out at 1-10 ug/mL, depending on the target cells’ sensitivity.

Since the production and application of lentiviral and retroviral vectors contain many steps, there are several points at which production or transduction efficiency may be hindered. Here we will discuss some of the most common issues:

A potential cause of this is that the packaging cells have been maintained at confluency, which results in a short term decrease in virus production. Consider thawing a fresh batch of cells. Alternatively, in the case of retroviruses, the phoenix cells may need to be doubly selected with hygromycin B and diphtheria toxin to ensure that all of the cells express both packaging constructs.

The ratio of the transfer and helper plasmids may need to be optimized for your particular system. Most companies will recommend a ratio to start with, but this may need to be adjusted.

While lentiviral and retroviral vectors can accommodate relatively large inserts as compared to other viral vector systems, there is still a packaging limit. Viral particles can accommodate 8-10 kb between the two LTRs. Transgene constructs longer than this will greatly decrease packaging efficiency, which would result in a lower viral titer.

Another possible way to overcome low titers is to concentrate the virus. After purification to remove the cell debris, the supernatant can be pelleted by ultracentrifugation. The pelleted virus can then be resuspended in the desired volume of media or buffer. Many companies provide products and reagents for virus concentration (see Reagents below).

If the yield of transduced cells is low despite a high titer of virus, it is possible that the total volume of transducing media on the target cells is too high [54]. The transduction step can be carried out in a volume of media that just covers the cells; this increases the exposure of the cells to the virus, and maximizes the likelihood of virus-cell interactions. Care should be taken to not dry out areas of the dish, for example by rocking the dish periodically during transduction. The volume can be brought up again after 4-6 hours to prevent the cells from drying out overnight.

It is also possible that the virus was collected too early. The peak of virus production is between 48-72 hours post-transfection.

Some cell types, such as primary fibroblasts or neuronal cells, are inherently difficult to transduce. This can be a difficult problem to solve, and may require either optimization of the transduction protocol or transducing with an increased titer. Pseudotyping of the transducing virus to target more abundant receptors on a particular cell type is another approach to consider.

Packaging cell medium may not be compatible with target cell growth. Either dilute the virus in target cell-compatible medium or concentrate it as described above and resuspend it in a medium compatible with the target cells.

There are many different companies that provide reagents for the production of lentiviral and retroviral vectors. When choosing what you will need, there are several considerations:

• How difficult is the insert you are trying to clone?
• Are your target cells difficult to transduce?
• Will you need high viral titers?
• What type of insert are you going to express?

Many of the reagents and transfer plasmids have been optimized for certain conditions (production of high viral titers) or designed with certain features (such as specific, dual, or inducible promoters). A commonly used lentiviral expression vector is Takara Bio / Clonetech pLVX-Puro with the constitutive CMV promoter [55].

There are several variations on the HEK-293 based packaging cells described above that are available from commercial sources:

Transfection reagents can be acquired commercially or formulated in the lab. In addition to the most common transfection reagents: lipofectamine [59, 60], calcium phosphate [44, 61], fugene [33], etc. there are also many commercially formulated kits that contain the transfer and helper plasmids and tout a higher virus yield and an optimized ratio of plasmids. The parameters for calcium phosphate transfection have been optimized [62, 63], and prolonged incubation with calcium phosphate might be toxic.

The following section discusses some common transfection reagents. Specialized transfection reagent kits also exist, for example, Clontech/Takara Bio Xfect mESC Transfection Reagent for mouse embryonic stem cell transfection [64], Jetprime [65, 66] or jetPEI [67] from Polyplus or TransIT-LT1 [68] or TransIT X2 [41] transfection reagent from Mirus .

Lipofectamine is a cationic lipid with a positively charged head group and 1-2 hydrocarbon chain. The head group interacts with the phosphate backbone of the nucleic acid. The positive surface charge of the liposomes allows for the fusion of liposome/nucleic acid complex with the negatively charged cell. As an example, Vodnala SK et al transfected Platinum-E ecotropic (PlatE) packaging cells from Cell Biolabs and pCL-Eco plasmid using Lipofectamine in OptiMEM from Invitrogen [58]. PLUS reagent from Thermo Fisher can improve the efficiency of transfection reagents, such as Lipofectamine [60].

Fugene is a nonliposomal transfection reagent that claims to have a high efficiency of transfection with low toxicity. It is compatible with serum and does not require changing of the medium after use. It is a proprietary mixture of positively charged lipids and other components that interact with the negatively charged DNA and allow for entry into the cell. Recent applications include [33, 70].

Note: When combining Fugene and plasmids, it is suggested that polystyrene tubes be used and the Fugene be added last to avoid it interacting with the tube.

Roche describes this as a “multi-component reagent that forms a complex with DNA, then transports the complex into animal or insect cells.” It allows for the transfection of DNA into a wide variety of cells with minimal toxicity. It has been cited in the literature [34].

Transfer plasmids are derived from various backbones. Typical vectors contain some, or all, of the following features which make the vectors safer, increase viral titers, or enhance expression of the insert.

MilliporeSigma offers many backbones (for example, pLKO.5 [71] ) for their MISSION shRNA plasmids. They have a wide range of fluorescent or drug marker options, and inducible systems. shRNA vectors employ a U6 promoter. They offer both positive and negative controls for all of the backbones. This was developed in collaboration with the RNA consortium scientists, and the pLKO.1-puro base vector was developed at the Broad Institute [72]. All of these are lentiviral vectors. Various MISSION shRNA plasmids sets and controls have been published in >1,500 journal articles.

SBI offers lentiviral vectors that are HIV- and FIV-based and have many promoters, depending on the cell type being transduced: CMV (Cytomegalovirus) for HeLa and many other cell types and for HEK293 and HT1080 transduction; MSCV (Murine Stem Cell Virus), for hematopoietic and stem cell transduction; UbC (Ubiquitin C), for transducing most cell types; PGK (Phosphoglycerate Kinase), for transducing most cell types; and EF1 (Elongation Factor 1α), for transducing most cell types [73] In addition to the transfer vectors listed below, they offer inducible and dual promoter backbones. Available selectable markers include GFP, RFP, Puromycin, Hygromycin, Neomycin and Zeocin. R Garcia-Martin et al, for example, introduced murine pre-miRNA sequences into pre-adipocytes, hepatocytes and endothelial cells using System Biosciences pCDH-CMV-MCS-EF1α-GreenPuro Cloning and Expression Lentivector [74].

A summary of their Backbone-Promoter and markers is described below.

Invitrogen offers the ViraPower™ Lentiviral Expression System. The transfer plasmids are pLP or pLenti-based vectors and are accompanied by three packaging/helper plasmids (pLP1, pLP2, and pLP/VSVG) [75]. They have lentiviral systems optimized for gene expression under the control of CMV, UbC, CMV/TO, RSV, or EF-1α promoters. They include several lentiviral vectors for the creation of Lumio or V5 epitope-tagged proteins. One distinction of the Invitrogen systems is that they provide multiple options for cloning including Gateway (DEST™), TOPO®, and recombination-based (pLenti6/UbC/V5-DEST™) systems. The various pLenti-based plasmids have been published in >2,000 journal articles.

Retrovirus Production

Cell Biolabs offers a range retroviral expression vectors [76] in various background plasmids that are murine leukemia virus (MLV)-derived. They offer retroviral packaging cell lines (293RTV) and kits for virus purification and concentration: ViraBind™ Concentration and Purification kits or ViraBind™ PLUS Concentration and Purification Kits. Their pMXs, pMYs, and pMCs vectors have been shown to be effective in induced pluripotent stem cells. They offer several selectable markers: Hygromycin, Neomycin, Puromycin, Zeomycin, and GFP. The various backbone vectors have been published in >500 journal articles. For example, Yasuda S et al introduced RAD23B and its variants to RAD23B-KO cells with a pMX-Puro vector (Cell Biolabs) [77].

Lentivirus Production

Cell Biolabs, Inc. offers lentiviral expression vectors based on the pSMPUW-IRES and pSMPUW-U6 backbones into which a gene of interest can be directly cloned. They include the following selectable markers: Blasticidin, Hygromycin, Neomycin, Puromycin, and GFP. These transfer vectors carry a kanamycin resistance gene, WPRE, Multiple Cloning Site (MCS), cPPT, 3’ LTR, and a 5’ CMV/LTR [78]. These expression vectors have been referenced in 6 journal articles.

Addgene is a nonprofit repository for plasmids and offers a wide variety of lentiviral and retroviral transfer vectors. A few commonly used retroviral vectors are listed in table 9. Others from Addgene include pBMN-PIB [57].

Popular lentiviral vectors available through Addgene are included in table 10. Lentiviral packaging plasmids PAX2 ( 12260) and pMD2.G ( 12259) from Addgene are very commonly used [41, 79]. Duncan A et al, for example, obtained p-Lenti-7xTcf-FFluc-SV40-mCherry from Addgene ( 24307) [80].

SBI System Biosciences offers a low-speed centrifugation alternative for retrovirus concentration with their Retro-Concentrin™ which precipitates the viruses followed by low-speed centrifugation. The process takes at least 12 hours. One advantage, however, is that the resulting pellet is stabilized for storage at -80 °C [82].

SBI System Biosciences also offers the Peg-it™ virus precipitation solution [33]. This solution is formulated with polyethylene glycol and has been optimized for lentiviral particle precipitation. When mixed with the media collected from the packaging cells, it causes the viral particles to precipitate. The mixture can then be centrifuged to pellet the particles. It increased the concentrations 10- to 100-fold.

Cell Biolabs, Inc. provides lentivirus concentration and purification kits that are either column-, dialysis-, or filter-based. The column-based kit allows for concentration up to 500 fold with higher purity that ultracentrifugation in 3-5 hours. These can process larger volumes than filter-based methods. The Dialysis-based kit allows for concentration up to 10⁹ TU/mL, with higher purity, in about 12-24 hours. Filter-based kits recover greater than 90 % of the virus with high quality in less than two hours.

GeneCopoeia offers the Lenti-Pac™ Lentivirus Concentration Solution which, through the use of its proprietary reagent, allows for a 10-100 fold increase in virus concentration in less than 3 hours without ultracentrifugation.

Other companies provide similar products as well. Lenti-X concentrator from Clontech (631231) is a popular choice for concentrating lentivirus [60, 83].

Cell Biolabs, Inc. provides a lentivirus quantitation/titering kit. This measures the viral nucleic acid content of either purified lentivirus or unpurified supernatant in 45-60 minutes. The virus is captured by beads and then denatured, and absorbance is read and compared to a standard curve (Lentivirus RNA Standard provided with the kit) to determine nucleic acid content.

They also provide another kit (QuickTiter™ Lentivirus Titer Kit) that measures the virus-associated p24 matrix protein by ELISA on an anti-p24 antibody-coated plate. A p24 antigen standard is provided for quantification. Fanning S et al used this kit to determine the titers of pLV-hSyn-hSNC and pLV-hSyn-mGFP expression vectors [84].

GeneCopoeia offers qRT-PCR based titering kits (Lenti-Pac™ HIV and FIV qRT-PCR Lentivirus Titration Kits). They provide the standard controls, RNA extraction reagents, and qRT-PCR reagents. The lentiviral RNA genome is quantified by qPCR using SYBR green technology.

Cell Biolabs, Inc. offers kits (ViraDuctin™ Lentivirus Transduction Kit and ViraDuctin™ Retrovirus Transduction kit) that comprise a proprietary cocktail that forms a supercomplex with the lentivirus particles, resulting in a 2- to 6-fold increase in transduction efficiency when compared to polybrene.

SBI System Biosciences offers TransDux™, which is an infection reagent with minimal toxicity that provides substantially increased transduction efficiency over polybrene.

They also offer a LentiMag™ kit which contains a formulation of magnetic nanoparticles that were developed for the transduction of mammalian cells with retrovirus or lentivirus vectors. By running a magnet under the cells being transduced, the magnetic nanoparticle-associated virus becomes concentrated onto the cells very rapidly, increasing the transduction efficiency.

Many of the companies listed above and others also offer ready-to-use viral transducing particles that have are high titer and purity (AddGene, MilliporeSigma; SBI System Biosciences; and others). For example, De Cecco M et al used pLKO-RB1-shRNA63 and pLKO-RB1-shRNA19 from T. Waldman through AddGene ( 25641 and 25640) [85]. Duncan A et al obtained pGF-CREB-mCMV-EF1α-Puro CREB reporter and the control vector in a lentivirus backbone from System Biosciences (TR202va-p) [80]. SE Sillivan et al obtained pre-miR-135b-5p and control in the CD513 lentiviral vector from System Biosciences [67].

Dana-Farber Cancer Institute and The Broad Institute provide the CCSB-Broad Lentiviral Expression Library for over 15,000 human ORFs in an expression-ready lentiviral system with a C-terminal V5 tag. Ebright RY et al infected isolated circulating tumor cells with several constructs from the library [60].

Despite the certain progress in current applications of lentivirus-based approaches, enhancement of gene transfer techniques remains the primary goal for the development of more efficient therapeutic protocols. Several studies have reported different methods to enhance transduction efficiency in hematopoietic stem cells (HSC). A recent study has demonstrated that UM171, an activator of HSCs significantly potentiated lentivirus-based transduction of CD34+ cells [86]. Also, according to Hauber et al, LentiBOOST, a membrane-sealing poloxamer, also enhances the efficiency of lentiviral transduction in human CD34+ cells obtained from peripheral blood [87] and murine T cells [52].

Also, a new platform for short-term gene expression using Integration-deficient lentiviruses (IdLVs), which express genes only transiently in dividing cells, has recently been introduced [88]. The authors have observed improved functional activity of HSCs when IdLVs have been used for delivery of HOXB4 and Angptl3. This technique helps avoiding the adverse effects found with constitutive expression.