Retroviral preparations containing albumin for enhancing transduction efficiency, uses thereof for cell transduction

Abstract

The invention relates to methods of enhancing the transduction of retroviridae into cells, methods of improving the efficacy of retroviridae based therapy, methods of producing cell therapy, and uses thereof In particular, the invention relates to a method of enhancing transduction of a retroviridae into a cell, the method comprising: (i) combining the retroviridae with albumin to form a pre-mix; and (ii) contacting the cell with the pre-mix formed in step (i).

Description

[0001] RETROVIRAL PREPARATIONS CONTAINING ALBUMIN FOR ENHANCING TRANSDUCTION

[0002] EFFICIENCY, USES THEREOF FOR CELL TRANSDUCTION

[0003] FIELD OF THE INVENTION

[0004] 5 The present invention relates to methods of enhancing the transduction of viruses, in particular retroviridae into cells, methods of improving the efficacy of retroviridae based therapy, methods of producing a cell therapy ex vivo, use of albumin to form a pre-mix for enhancing transduction of a retroviridae into a cell, and use of a pre-mix for enhancing transduction of a retroviridae into a cell.

[0005] BACKGROUND OF THE INVENTION

[0006] The family of retroviridae, including the subfamily orthoretrovirinae, are defined by the ability to reverse transcribe an RNA genome into DNA that integrates into the cell5 genome during the intracellular virus life cycle. The family of Retroviridae includes retroviruses.

[0007] Retroviruses contain a non-segmented RNA genome, and their hallmark is a replicative strategy, which includes reverse transcription of the virion RNA into linear double¬0 stranded DNA (also known as provirus) and the subsequent integration of this DNA into the host genome. In general, retroviruses enter cells through the attachment of their surface glycoproteins to specific cell membrane receptors, leading to fusion of virus and cell membranes. The provirus is transcribed into mRNAs that encode the viral proteins, which subsequently package the full-length genomic mRNA into virions5 to complete the virus life cycle.

[0008] Retroviruses comprise a large and diverse family of enveloped RNA viruses (referenced below). Retroviruses and lentiviruses belong to the retroviridae family. Lentiviruses are enveloped, single stranded positive sense RNA viruses (Clements & Zink, 1996). Lentiviruses can infect a range of human cells such as macrophages, lymphocytes, and monocytes. Unlike other retroviruses, lentiviruses can deliver genetic material to both dividing and non-dividing cells (Clements & Zink, 1996). As the lentivirus genome integrates into the host DNA, the lentiviral genome is passed onto the daughter cells of the host. There is the possibility that the lentivirus could mutate / recombine through5 generations resulting in an infectious replication competent lentivirus, which could put the patient and the scientists who manufacture the therapy at risk. Current lentiviral vectors are modified to reduce safety concerns. First generation lentiviral vectors had limitations because although the packaging signal within the vector was deleted, the packaging genes {gag and poi were together, meaning that only one homologous recombination event was required to reinstate a vector's replicative competence (Rodrigues et al., 2011). Modern methods however use third generation lentiviral vectors, which separate gag, pol and env on different plasmids (Escors & Breckpot, 2010), meaning at least 3 homologous recombination events are required to restore replication competence, which are unlikely to take place.

[0009] The ability of viruses to infect and genetically modify human cells can be utilised therapeutically in gene therapies, such as in the treatment of beta-thalassemia, haemophilia and adrenoleukodystrophy (Vargas et al., 2016). Retroviridae vector technology is the method used most commonly for gene transfer in gene therapy. There are two general approaches: namely, in vivo gene therapy, in which genes are delivered directly to target cells; and ex vivo therapy, in which the target cells are genetically modified outside the body and then transplanted into a subject.

[0010] In a cell modified gene therapy, lentivirus is commonly used as a vector to introduce a transgene into the cell, a process is known as transduction. Transduction is the process of virus-mediated transfer of nucleic acids into a cell. The virus is able to infect the cell and transport the nucleic acids into the nucleus, independent of further actions. Transduction can be transient, for example wherein the nucleic acids remain episomal or can be stable, for example wherein the nucleic acids integrate into the cell genome. For example, lentiviruses have the ability to permanently integrate into the genome of the cell. After the virus has entered the cell, the viral RNA is transcribed by the reverse transcriptase to produce double-stranded DNA that enters the nucleus. The transgene is integrated into the host genome via the lentiviral integrase enzymes. Once a cell has been transduced by a lentiviral particle, the cell expresses the transcript and / or protein of a gene of interest.

[0011] Although several clinical trials for retroviridae-based therapies are under way, success has been limited. A key issue faced in retroviridae-based therapies is that a high multiplicity of infection (MOI) of viral particles is needed to provide sufficient integration and expression of the transgene. Multiple avenues are currently used to help increase transduction of viruses, for example: increasing viral titer to increase the multiplicity of infection, prolonging viral infection duration, and / or repeating multiple rounds of viral infection. This can lead to increased side effects and risks from off target effects.

[0012] Other avenues to ensure sufficient expression of the transgene include overexpressing viral receptors (e.g., PIT1 and PIT2) on the cells to be transduced and / or optimising the viral vector cassette by using a strong gene promoter and / or gene enhancer, codon optimisation of the transgenic cDNA, effective polyadenylation sequence and the use of a self-complementary vector genome. However, such methods are not generally amenable to all viral vectors and / or transgenes.

[0013] Transduction enhancers are commonly used to aid transduction. These work by assisting the virus in transduction resulting in a higher proportion of cells being transduced or even enabling the MOI to be reduced while maintaining the same level of transduction. Polybrene is a retroviridae transduction enhancer that, as a polycation, is suggested to work to neutralise the electrostatic repulsion between the surface of the cellular and viral lipid bilayers and promote the binding of the virus to the cell surface (Andreadis & Palsson, 1997). Polybrene is inexpensive and enhances transduction efficiency (Davis et al., 2002), so it is a popular choice of enhancer; however, it can be toxic to cells if used at high concentrations. Another transduction enhancer is known as Lentiboost®. This contains poloxamer 338 which can be defined as a 'large non-ionic amphiphilic molecule' (Lee et al., 1992) that is suggested to increase transduction by interaction with the cell membrane to allow the passage of genetic material (Lee et al., 1992); however, this commercial product is expensive.

[0014] Consequently, there is an unmet need for enhancers of lentivirus transduction that are safer and / or less expensive.

[0015] Albumin is a key protein found in blood and makes up 60% of the total protein content of serum (Francis, 2010). Albumin is a heart shaped polypeptide molecule with a molecular mass of 66.5 kDa (Sand et al., 2014). It is a non-glycosylated helical molecule and is made up of 585 amino acids (Horvathy et al., 2017). Albumin has many biological functions; a notable function is that albumin is an antioxidant which means it is useful in reducing oxidative stress within a cell. This is due to reduction of the free thiol group found on the Cysteine-34 residue, meaning albumin can scavenge free radicals (Francis, 2010). Albumin is also known to directly bind in a nonspecific manner to the surface of a cell to 'protect a cell', which makes it useful biomedically (Yamazoe and Tanabe, 2008). Due to this, albumin can be useful in medical devices to prevent the formation of biofilms. Another key function of albumin is that it can bind to fatty acids, lipids and metal ions to help influence cellular processes and is used as a key component in cell culture growth media. Fetal bovine serum (FBS) is the standard addition to cell culture media, however human serum albumin (HSA) has become a popular choice due to the demand for animal component-free media in the cell and gene therapy space. Moreover, researchers are switching to recombinant sources of albumin due to supply chain issues and regional variations in HSA.

[0016] Wang et al. (2017), found that if Adeno Associated Virus (AAV) is pre-incubated with HSA, the transduction efficiency of the virus is increased. The improvement in transduction efficiency found by Wang et al. (2017) was suggested to work by direct interaction between albumin and the AAV capsid. However, lentivirus differs structurally from AAV due to the presence of a lipid envelope which surrounds the lentivirus capsid. Therefore, it was not anticipated that albumin would be able to enhance lentiviral transduction, as was achieved with AAV. Surprisingly and unexpectedly, however, the present inventors have demonstrated that albumin can enhance lentiviral transduction in at least human embryonic kidney (HEK)293 cells and Jurkat cells. Specifically, pre-mixing albumin and lentivirus before addition to the cells was found to enhance transduction of the lentivirus such that it was possible to reduce the MOI and maintain the same level of transduction. The present inventors also found that combining known transduction enhancers with albumin displayed at least a 2-fold increased transduction efficiency ( / .e. at least a doubling) compared to using the transduction enhancer alone.

[0017] SUMMARY OF THE INVENTION

[0018] The invention relates to a method of enhancing transduction of a retroviridae into a cell, the method comprising: (i) combining the retroviridae with albumin to form a premix; and (ii) contacting the cell with the pre-mix formed in step (i).

[0019] The invention also relates to a method of improving the efficacy of a retroviridae based therapy, the method comprising combining the retroviridae with albumin to form a premix; and administering the pre-mix to a subject.

[0020] The invention also relates to a method of producing a cell therapy ex vivo, wherein the cell therapy comprises cells transduced by a retroviridae, the method comprising: (i) providing the cells to be transduced by the retroviridae, and (ii) carrying out the method of Claim 1 on the cells ex vivo so as to produce the cell therapy.

[0021] The invention also relates to a retroviridae based cell therapy for use in treating a subject.

[0022] The invention also relates to a method of treating a subject comprising administering a retroviridae based cell therapy to a subject in need thereof, wherein the method comprises (a) forming the pre-mix; (b) adding the pre-mix to the cell; and (c) administering the transduced cell to the subject.

[0023] The invention further relates to a retroviridae based therapy for use in treating a subject, wherein the retroviridae is combined with albumin to form a pre-mix, which is subsequently administered to the subject.

[0024] The invention also relates to a method of treating a subject comprising administering the retroviridae based therapy to a subject in need thereof, wherein the method comprises (a) forming the pre-mix; and (b) administering the pre-mix to the subject.

[0025] The invention also relates to the use of albumin to form a pre-mix for enhancing transduction of a retroviridae into a cell, wherein the albumin is combined with the retroviridae to form the pre-mix before contacting the cell.

[0026] The invention further relates to the use of a pre-mix for enhancing transduction of a retroviridae into a cell, wherein the pre-mix comprises albumin and the retroviridae and is formed before contacting the cell.

[0027] BRIEF DESCRIPTION OF THE FIGURES

[0028] Figure 1 - The impact a pre-mix of albumin and lentivirus has on the transduction efficiency of (A) Jurkat cells (B) HEK293 cells. For the pre-mix, albumin and lentivirus were both added to an Eppendorf tube and vortexed for a few moments, e.g. for 2-3 seconds. For 'Albumin then Lenti', albumin was pipetted into the well, followed by lentivirus. For 'Lenti then Albumin', lentivirus was pipetted into the well followed by albumin. For 'Together but separate', both albumin and lentivirus were pipetted into the well at the same time. Transduction efficiency was measured by % GFP detected by the FITC channel on the FACS Celesta Flow Cytometer.

[0029] Figure 2 - The effect that the length of time a pre-mix is incubated has on transduction efficiency of (A) Jurkat cells and (B) HEK293 cells. The incubations took place at room temperature and range from immediately (within seconds) to 24 hours before the premixes were added to cells.

[0030] Figure 3 - Different Concentrations of albumin (Recombumin® Elite) were investigated in the pre-mix of albumin and lentivirus to determine the optimal concentration / range of concentrations that provided the greatest transduction efficiency in (A) Jurkat cells and (B) HEK293 cells. Albumin concentrations refer to the stock albumin concentration. Refer to Example 1 for final concentration of albumin in the pre-mix.

[0031] Figure 4 - Effect of Different Sources of Albumin on Transduction. Different recombinant albumins and human serum albumins were used in the pre-mix at a concentration of 25 mg / mL (upon dilution with Lentivirus, the final concentrations of albumin in the pre-mix were 17.8 mg / mL and 22.3 mg / mL for Jurkat cells and HEK293 cells respectively) and then utilised to transduce cells (A) Jurkat cells and (B) HEK293 cells.

[0032] Figure 5 - Comparison of Alternative Sources (animal species sequences) of Albumin on Transduction. Different albumins were used in the pre-mix at a concentration of 25 mg / mL and then utilised to transduce cells (A) Jurkat cells and (B) HEK293 cells.

[0033] Figure 6 - Effect of MOI on transduction enhancement. A range of MOI's were examined to determine if the effect of pre-mixing the lentivirus with albumin (Recombumin® Elite) still enhanced transduction with different amounts of lentivirus, (A) Jurkat cells and (B) HEK293 cells.

[0034] Figure 7 - Effect of combining commercially available transduction enhancers and albumin (Recombumin® Elite) pre-mix. A and B show the data obtained with the transduction enhancer Polybrene for Jurkat and HEK293 cells respectively. C and D show the data obtained with the transduction enhancer Lentiboost® for Jurkat and HEK293 cells respectively.

[0035] Figure 8 - Effect of freezing and storing the albumin (Recombumin® Elite) pre-mix. Pre-mix samples were prepared at time 0, aliquoted and either utilised immediately in a transduction assay or stored at -80°C and used monthly in a transduction assay for a period of 3 months transductions were performed in HEK293 cells.

[0036] Figure 9 - Effect on transduction efficiency when storing the lentivirus pre-mix with albumin (Recombumin® Elite) at room temperature before transducing the (A) Jurkat cells and (B) HEK293 cells. Number indicates the fold increase in transduction of Recombumin Elite pre-mix compared to PBS pre-mix.

[0037] Figure 10 - Effect on transduction efficiency of storing the lentivirus in PBS at room temperature before combining with albumin (Recombumin® Elite) to form the premix and immediately adding to (A) Jurkat cells and (B) HEK293 cells. Number indicated the fold increase in transduction of Recombumin Elite premix compared to PBS pre-mix. The fold increase is not statistically significant for 4-10hrs for Jurkat cells and 4-8hrs for HEK293 cells.

[0038] Figure 11 - The effect of different concentrations of albumin on the transduction enhancement of lentivirus into Jurkat cells (A) and HEK293 cells (B). Albumin concentrations refer to the stock albumin concentration. Refer to Example 2 for final concentration of albumin in the pre-mix.

[0039] Figure 12 - The use of albumin ("Sacch.") in the enhancement of lentiviral transduction of T cells (A) and BM-MSC (B). The graphs also show the effect of the commercially available transduction enhancer Lentiboost® alone and in combination with the albumin.

[0040] Figure 13 - The use of albumin ("Elite") in the enhancement of lentiviral transduction of CD 34+ cells.

[0041] DETAILED DESCRIPTION OF THE INVENTION

[0042] In a first aspect, the invention provides a method of enhancing transduction of a retroviridae into a cell, the method comprising: (i) combining the retroviridae with albumin to form a pre-mix; and (ii) contacting the cell with the pre-mix formed in step (i).

[0043] By "enhancing", "enhancement of" or "enhanced" transduction, we include the meaning that the ability of the virus to transduce a cell is increased, for example relative to the ability of the virus to transduce a cell when the virus has not been pre-mixed with albumin. We also include the meaning that the transduction efficiency is increased.

[0044] Methods of assessing the ability of a virus to transduce a cell are well known in the art and any suitable method can be used. Examples of methods are described below and in the Examples.

[0045] Conveniently, the virus contains a detectable nucleic acid in its genome, which nucleic acid can be detected inside the cell once the virus has transduced the cell. The detectable nucleic acid could be a nucleic acid that is detectable directly. For example, the nucleic acid may be detected following hybridisation with a probe that comprises a detectable moiety (e.g. a fluorophore or radiolabel). The nucleic acid may be detected indirectly, for example following expression of a protein encoded by the nucleic acid which protein can be detected, for example by virtue of assessing its expression or activity. It will be appreciated that the genome of the virus may be modified to include the detectable nucleic acid which can serve as a so-called "reporter gene" to measure the level of transduction. In this work, the reporter gene green fluorescent protein (GFP) was utilised as the transgene, and GFP expression in a cell was monitored to determine the levels of transduction enhancement. Other suitable reporter genes are known in the art and include yellow fluorescent protein, lacZ, alkaline phosphatase, and firefly luciferase.

[0046] In one embodiment, the transduction of the retroviridae into a cell is enhanced at least 1.01 fold compared to the transduction of a retroviridae into a cell when the retroviridae is not pre-mixed with albumin, for example at least 1.02, 1.03, 1.04, 1.05,

[0047] 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,

[0048] 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,

[0049] 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,

[0050] 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,

[0051] 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10- fold compared to the transduction of a retroviridae into a cell when the retroviridae is not pre-mixed with albumin. By "fold change", we include the meaning of the change in the percentage of a population of cells that are successfully transduced, with "1- fold", we include the meaning of no change in the percentage of a population of cells that are successfully transduced, and (for example) a 1.05-fold enhancement meaning a 5% increase in the percentage of a population of cells that are successfully transduced.

[0052] In one embodiment, the enhanced transduction is manifest by an increase in the expression of a transgene inside the transduced cell, optionally wherein the increase in the expression of the transgene is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,

[0053] 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5,

[0054] 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,

[0055] 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1,

[0056] 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10 times compared to the expression of a transgene into a cell when the retroviridae is not pre-mixed with albumin. As an example, in this context, an increase in expression of "1." times" means a 50% increase. By "transgene", we include the meaning of an exogenous nucleic acid which is incorporated into the genome of the retroviridae. For example, a transgene could be a reporter, a marker, and / or a therapeutic transgene. However, it is appreciated that the transgene need not include a reporter or marker.

[0057] The transgene may comprise any gene of interest, e.g. a nucleic acid sequence that encodes a protein that is desirable for integration into the genome of the retroviridae. Expression of a transgene may be transient or stable / integ rated. Conveniently, the gene of interest may be operably linked to one or more other sequences that are useful for obtaining the desired expression of the gene of interest, such as transcriptional regulatory sequences (e.g. promoters, enhancers, terminators, post transcriptional regulatory elements (PREs)). An example of a PRE is woodchuck hepatitis virus PRE (WRPE). Preferably, the transgene is under the control of a promoter. Suitable promoters include housekeeping or ubiquitous promoters such as CMV, PGK, EF-lol, MND, MCU3, SFFV and CBh) or tissue specific promoters (such as CDllb, ALB, TBG, MHO, MLC2v and eTnT promoters). In the context of a therapeutic transgene, tissue specific promoters may be used to ensure that the level of gene expression is in line with the expression of the "damaged" gene or the absent gene that the transgene is intended to replace / augment.

[0058] In one embodiment, the transgene encodes a protein reporter. The protein reporter may be a fluorescent protein (such as GFP, dsRed, or mCherry), a luminescent protein (such as luciferase or any other bioluminescent or chemiluminescent molecule), an enzymatic reporter (such as LacZ (P-galactosidase)), an antibiotic resistance marker (such as a puromycin resistance marker) or a combination thereof.

[0059] It will be appreciated that the transgene may encode more than one gene of interest. For example, the transgene may encode a therapeutic protein and a reporter and / or marker protein. The presence of a reporter or marker may be useful to monitor or detect expression of a therapeutic protein in a subject. However, it is also recognised that the expression of therapeutic proteins can be detected without the use of reporter or marker molecules, and so it is not necessary for the transgene to also comprise a reporter or marker.

[0060] In one embodiment, the enhanced transduction is manifest by an increase in the expression of one or more RNA species inside the transduced cell. Examples of RNA species may include mRNAs, short interfering RNAs, small hairpin RNAs, short guide RNA, long noncoding RNAs or microRNAs. By "expression" of a transgene or RIMA species, we include the meaning of one or more of the following events: (1) reverse transcription of the viral RNA into DNA (2) integration of the viral DNA into the host genome (3) production of an RNA template from a DNA sequence (e.g., by transcription); (4) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (5) translation of an RNA into a polypeptide or protein; (6) folding of a polypeptide or protein; and (7) post- translational modification of a polypeptide or protein.

[0061] Those skilled in the art would be capable of measuring the expression of a transgene. For example, RNA templates or transcripts can be detected e.g. using polymerase chain reaction and quantified e.g. using Real time (RT) quantitative PCR (qPCR), quantitative reverse transcription PCR, digital PCR or digital drop PCR; polypeptides or proteins can be detected e.g. using immunohistochemistry and quantified e.g. using flow cytometry, Western blots or enzyme-linked immunosorbent assay (ELISA).

[0062] In another embodiment, the enhanced transduction is manifest by an increase in the activity of a protein encoded by a transgene inside the transduced cell.

[0063] It will be appreciated that different proteins have different functions, for example: enzymes {e.g., amylase, lipase, pepsin), transport {e.g., hemoglobin), structural support {e.g., actin, tubulin, keratin), chemical signalling {e.g., insulin), and immune response {e.g., antibodies). By "activity of a protein", we include the meaning that the protein is performing its known function. By "increase" in activity, we include the meaning that the protein is performing its known function more optimally or at a higher level. For example, enzymes may process more substrate into product, which increase in product may be detected, and so on.

[0064] Those skilled in the art would be capable of measuring transduction of a retroviridae into a cell using any suitable method. For example, retroviridae may contain reporters or markers (such as fluorescent markers or antibiotic resistance markers). The number of fluorescent cells or antibiotic resistant cells in the population can then be quantified. For example, flow cytometry with a fluorescent protein marker {e.g., fluorescence- activated cell sorting (FACS)) or antibiotic selection can be used to determine the percentage of infected cells. In a particular example, retroviridae particles may comprise a gene encoding a reporter or marker (e.g., fluorescent protein) under the control of a promoter, such that when the retroviridae is transduced into a cell, the gene is expressed, and the reporter or marker (e.g., a fluorescent protein) is produced. A plate reader can be used to measure bioluminescence of a luminescent marker. A live cell imager (such as an Incucyte® live-cell analysis system, Sartorius) can be used to measure a fluorescent protein.

[0065] The level of transduction of a virus can be expressed as a transduction efficiency, namely the proportion of cells that a virus successfully transduces once the virus comes into contact with a population of cells. Thus, by enhancing transduction of a virus we include the meaning that the transduction efficiency of the virus is increased, for example relating to the transduction efficiency of the virus when the virus has not been pre-mixed with albumin.

[0066] By "pre-mix", we include the meaning of a composition comprising retroviridae and albumin. The retroviridae and albumin components of this pre-mix are mixed together prior to contacting the cell to be transduced, and so it will be appreciated that the composition does not include the cell to be transduced. Typically, therefore, the premix is cell-free. By "cell-free", we include the meaning that the composition is substantially free of cells, in particular the cells to be transduced. Mixing may or may not include agitation such as rocking, shaking, flicking, oscillating in a generally circular motion e.g. vortexing, or repetitive pipetting (i.e., repeatedly dispensing and withdrawing solution by pipetting, typically up and down). Preferably, the pre-mix is formed by oscillating, e.g. vortexing, for 1-4 seconds, e.g. 2-3 seconds.

[0067] In a preferred embodiment, step (ii) is performed immediately after step (i). By "immediately after", we include the meaning that the cells are contacted with the premix within 3, 5, 10, 15, 20, 25, 30 seconds of combining the retroviridae with the albumin to form the pre-mix. For example, step (ii) may be performed within 1-30 seconds of combining the retroviridae with the albumin. Preferably, step (ii) is performed immediately after step (i) or within a timeframe of about 0 (immediately) to about 2 hours, such as from about 1, 5, 10, 20 or 30 seconds to about 10, 20, 30, 40, 50, 60 minutes.

[0068] In another embodiment, step (ii) is performed within a timeframe of from about 30 seconds to about 36 hours after step (i), such as within a timeframe of from about 30, 35, 40, 45, 50, 55 or 60 seconds to about 1, 2, 4, 6, 8, 12, 18, 24, 28, 32, 33, 34, 35 or 36 hours, such as within a timeframe of about 1, 2, 3, 4, 5, 10, 15, 30, 45 or 60 minutes. In one embodiment, step (ii) is performed not more than about 30 seconds after step (i), or not more than about 5 minutes after step (i), such as not more than about 10, 20, 30, 40, 50 or 60 seconds, not more than about 1, 2, 3, 4 or 5 minutes.

[0069] In one embodiment, the pre-mix formed in step (i) is stored at a temperature of from about 0 to about 35 degrees centigrade (°C), or at room temperature, before contacting the cell in step (ii), such as from about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20°C to about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35°C.

[0070] By "stored", we include the meaning of incubating the pre-mix at a non-frozen temperature, such as from 0°C to 35°C, for any period of time provided that the retroviridae is still capable of being transduced inside the cell. By "room temperature", we include the meaning of a temperature of about 21°C.

[0071] In one embodiment, the pre-mix formed in step (i) is stored at a temperature of from about 15 to about 35°C, or at room temperature, such as from about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 °C to about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35°C for a period of from about 30 seconds to about 36 hours (e.g. from about 30 seconds to about 24 hours), such as a period of from about 30, 35, 40, 45, 50, 55 or 60 seconds, 1, 2, 5, 10, 15 minutes to about 1, 2, 4, 6, 8, 12, 18, 24, 28, 32, 33, 34, 35 or 36 hours before contacting the cell in step (ii).

[0072] In another embodiment, the pre-mix formed in step (i) may be stored at a temperature of from at least 35°C to about 47°C, such as from 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5 or 40 to 39, 40, 41, 42, 43, 44, 45, 46 or 47°C for a period of from 30 seconds to 8 hours, such as a period of from 30, 35, 40, 45, 50, 55 or 60 seconds, 1,

[0073] 2, 5, 10, 15 minutes to about 1, 2, 5, 10, 15, 30, 45, 60 minutes, 1, 2, 4, 6, 8 hours before contacting the cell in step (ii).

[0074] In a further embodiment, the pre-mix formed in step (i) is stored at a temperature of from about 0 to about 5°C, such as from about 0, 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0°C to about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5°C for a period of from about 30 seconds to about 72 hours, such as a period of from about 30, 35, 40, 45, 50, 55 or 60 seconds, 1, 2, 5, 10, 15, 30, 45 or 60 minutes, 1, 2 or 3 hours to about 30, 45, 60 minutes, or 1, 2,

[0075] 3, 4, 5, 6, 8, 12, 18, 24, 36, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 67, 68, 69, 70, 71, 72 hours before contacting the cell in step (ii). In a preferred embodiment, steps (i) and (ii) are carried out at room temperature, and step (ii) is performed immediately after step (i).

[0076] As shown in Example 1, the inventors have shown that the enhancement in transduction is still apparent when the pre-mix is formed in step (i) and immediately frozen, and then subsequently thawed before carrying out step (ii). Thus, in one embodiment, the pre-mix is frozen {e.g., at from about -15°C to about -90°C, e.g., at from about -15°C to about -25°C , e.g., at from about -70°C to about -90°C, e.g., at about -20°C or about -80°C) and thawed (e.g., from about 2°C to about 6°C, e.g., at about 4°C or at about room temperature) before being contacted with cells in step (ii). The pre-mix may be frozen (e.g., at from about -70°C to about -90°C, e.g., at about -80°C) for at least one day (e.g., at least 2, 3, 4, 5 or 6 days), or at least one week (e.g., at least 2, 3 or 4 weeks), or at least one month (e.g., at least 2 or 3 months, or at least 4, 5 or 6 months) before being thawed and contacted with cells in step (ii).

[0077] In one embodiment, step (i) further comprises freeze-thaw cycles. By 'freeze-thaw cycles', we include the meaning of cycles where the formulation is frozen at from about -15°C to about -90°C, e.g., at from about -15°C to about -25°C , or e.g., at from about -70°C to about -90°C, preferably at around -20°C or at around -80°C for a set period (e.g., at least 19 hours) and thawed at room temperature for a set period (e.g., at least 5 hours) before being frozen again and repeating the cycle.

[0078] The cell may be any cell that is known to be transduced or capable of being transduced with the retroviridae (e.g., the retrovirus or lentivirus). In one embodiment, the cell is a eukaryotic cell. Preferably the cell is an animal cell, a fungal cell, such as a yeast cell, or a plant cell. In one embodiment, the animal cell is a mammalian cell, a fish cell, an insect cell, a reptile cell, an amphibian cell or an avian cell.

[0079] Different subclasses of the retroviridae family may have different abilities to transduce different types of cells. For example, lentiviruses are capable of transducing dividing cells and non-dividing cells, whereas another subclass of the retroviridae family, y- retroviruses, may only transduce dividing cells. This is because they are only able to enter the nucleus during mitotic breakdown of the nuclear envelope.

[0080] Hence, it will be appreciated that the cell may be a dividing cell or a non-dividing cell. By a "dividing cell", we include the meaning of a cell that is or (in its current state) is capable of undergoing cell division, such as mitosis or meiosis. Such dividing cells can be totipotent or pluripotent and include stem cells, such as embryonic stem cells, adult stem cells or stem cell in plant meristems. Some cells (e.g., CD34 + stem cells) are 'slowly' dividing cells. By a "non-dividing cell", we include the meaning of a cell that is not undergoing cell division or (in its current state) is incapable of undergoing cell division, such as terminally differentiated cells. Such non-dividing cell can include myocytes, such as card io myocytes; primary neurons; glial cells; lymphocytes; macrophages; dendritic cells; epithelial cells; adipocytes.

[0081] Under certain conditions, a dividing cell may become a non-dividing cell by entering an inactive stage of mitosis (Go phase also known as the quiescent stage). Under certain conditions, a non-dividing cell may become a dividing cell.

[0082] In one embodiment, the cell is a cell line. By "cell line" we include the meaning of a defined population of cells that can be maintained in culture for an extended period of time, retaining stability of certain phenotypes and functions. Preferably, the cell line is a human embryonic kidney cell line, a T cell (e.g. a human or murine T cell), an immortalised CD4+ T cell line, such as a Jurkat cell line, a peripheral blood mononuclear cell (PBMC), a stem cell (e.g. a mesenchymal stem cell (MSC) or a hematopoietic stem cell), a neuron, a cardiomyocyte, a fibroblast, a Chinese hamster ovary (CHO) cell or a HMEC-1 cell.

[0083] In one embodiment, the cells are adherent cells, such as HEK293 cells, HT-1080 or HeLa cells. By "adherent", we include the meaning that the cells grow attached to the surface of a culture vessel.

[0084] In another embodiment, the cells are in suspension in the culture medium, such as Jurkat cells or T cells (e.g. a human or murine T cell; optionally, wherein the T cells originate from at least one donor).

[0085] In another embodiment, the cells are CD34+ cells. Examples of CD34+ cells can include but are not limited to hematopoietic stem cells, early hematopoietic progenitor cells, and B cells.

[0086] In another embodiment, when the cells are contacted with the pre-mix in vivo, the cell could be neuronal cell, a glial cell, an immune cell, a non-activated T cell, a thymus cell, a B cell, a myeloid cell or a lymphoid cell.

[0087] The term "albumin" as described herein includes the meaning of a protein having the same and / or very similar amino acid sequence and / or tertiary structure as human serum albumin (in particular SEQ ID NO: 1 which is the mature sequence provided by UniProt P02768), HSA domains and may have similar properties to HSA or the relevant domains. Similar tertiary structures are, for example, the structures of the albumins from species other than human, e.g., non-human primate albumin, (such as chimpanzee albumin e.g., predicted sequence GenBank XP_517233.2), gorilla albumin or macaque albumin {e.g., GenBank NP_001182578)), rodent albumin (such as hamster albumin {e.g., GenBank A6YF56), guinea pig albumin {e.g., UniProt Q6WDN9-1), mouse albumin {e.g., GenBank AAH49971, mature form being residues 25 to 608) or UniProt P07724-1 Version 3) and rat albumin {e.g., GenBank AAH85359 or UniProt P02770-1 Version 2)), bovine albumin (such as cow albumin {e.g., UniProt P02769-1, mature sequence being residues 25 to 607)), equine albumin (such as horse albumin {e.g., UniProt P35747-1) or donkey albumin {e.g., UniProt Q5XLE4-1), rabbit albumin {e.g., UniProt P49065-1 Version 2), goat albumin {e.g., GenBank ACF10391), sheep albumin {e.g., UniProt P14639-1), dog albumin {e.g., NCBI NP_001003026, mature form being residues 25 to 608), chicken albumin {e.g., UniProt P19121-1 Version 2) and pig albumin {e.g., UniProt P08835-1 Version 2), or any one of SEQ ID NOs: 4 to 19 of WO 2013 / 006675, which are incorporated herein by reference. All of these albumins are included in the scope of the present invention. Preferably, the albumin comprises an amino acid sequence of a mammalian albumin, such as a human albumin, bovine albumin, dog albumin or mouse albumin. Mature forms of albumin {e.g., forms where all post-translational modifications and / or processing steps have been completed such as SEQ ID NO: 1) are particularly preferred, and the skilled person is able to identify mature forms using publicly available information such as protein databanks and / or by using signal peptide recognition software such as SignalP {e.g., SignalP (Nielsen et al, 1997, Protein Engineering 10(1): 1-6)). SignalP Version 6.0 is preferred (Teufel et al, 2022, Nature Biotechnology 40: 1023-1025). An albumin preparation for use in the methods and compositions of the present invention may comprise one or more (several) albumins.

[0088] Some of the major properties of albumin are i) its ability to regulate plasma volume, ii) a long plasma half-life of around 19 days ± 5 days, iii) ligand binding, e.g., binding of endogenous molecules such as acidic, lipophilic compounds including bilirubin fatty acids, hemin and thyroxine (see also Table 1 of Kragh-Hansen et al, 2002, Biol Pharm Bull 25(6):695-704, hereby incorporated by reference), iv) binding of small organic compounds with acidic or electronegative features, e.g., drugs such as warfarin, diazepam, ibuprofen and paclitaxel (see also 1 of Kragh-Hansen et al, 2002, Biol Pharm Bull 25(6):695-704, hereby incorporated by reference). Not all of these properties need to be fulfilled in order to characterize a protein or fragment as an albumin. If a fragment, for example, does not comprise a domain responsible for binding certain ligands or organic compounds, the variant of such a fragment is not expected to have these properties either.

[0089] The term "albumin" includes fragments and / or variants. Thus, in one embodiment, the albumin is a wild-type albumin or a variant thereof. By "a variant thereof" we include the meaning of a polypeptide derived from a parent albumin comprising an alteration, i.e., a substitution, insertion and / or deletion, at one or more (several) positions. Such alterations may be referred to as "mutations". A substitution includes the meaning of a replacement of an amino acid occupying a position with a different amino acid; a deletion includes the meaning of the removal of an amino acid occupying a position; and an insertion includes the meaning of adding amino acids (e.g., 1-3 amino acids) adjacent to an amino acid occupying a position. For example, the altered polypeptide (variant) can be obtained through human intervention by modification of the polynucleotide sequence encoding the parent albumin.

[0090] In one embodiment, the albumin is a variant of a wild-type albumin, optionally wherein the variant albumin has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the wild-type albumin, such as at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with wild-type human albumin according to SEQ ID NO: 1 :

[0091] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDK SLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFH DNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA KQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRA DLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAK DVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIK QNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYL SVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEK ERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAAL GL

[0092] Preferably, the albumin has at least 70% sequence identity to a mammalian albumin, preferably from an albumin selected from the group consisting of human (SEQ ID NO: 1), mouse (SEQ ID NO: 2), dog (SEQ ID NO: 3), rat, macaque, bovine (SEQ ID NO: 4), pig, horse, rabbit, or guinea pig albumin. Mouse albumin sequence (SEQ ID NO: 2):

[0093] EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDK SLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKE NPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSV RQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDR AELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAE AKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNL VKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDY LSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPE KEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDA LA

[0094] Dog albumin sequence (SEQ ID NO: 3) :

[0095] EAYKSEIAHRYNDLGEEHFRGLVLVAFSQYLQQCPFEDHVKLAKEVTEFAKACAAEESGANCDK SLHTLFGDKLCTVASLRDKYGDMADCCEKQEPDRNECFLAHKDDNPGFPPLVAPEPDALCAAFQ DNEQLFLGKYLYEIARRHPYFYAPELLYYAQQYKGVFAECCQAADKAACLGPKIEALREKVLLSSA KERFKCASLQKFGDRAFKAWSVARLSQRFPKADFAEISKVVTDLTKVHKECCHGDLLECADDR ADLAKYMCENQDSISTKLKECCDKPVLEKSQCLAEVERDELPGDLPSLAADFVEDKEVCKNYQE AKDVFLGTFLYEYARRHPEYSVSLLLRLAKEYEATLEKCCATDDPPTCYAKVLDEFKPLVDEPQNL VKTNCELFEKLGEYGFQNALLVRYTKKAPQVSTPTLVEVSRKLGKVGTKCCKKPESERMSCAED FLSVVLNRLCVLHEKTPVSERVTKCCSESLVNRRPCFSGLEVDETYVPKEFNAETFTFHADLCTLP EAEKQVKKQTALVELLKHKPKATDEQLKTVMGDFGAFVEKCCAAENKEGCFSEEGPKLVAAAQA ALV

[0096] Bovine albumin sequence (SEQ ID NO: 4) :

[0097] DTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPFDEHVKLVNELTEFAKTCVADESHAGCEKS LHTLFGDELCKVASLRETYGDMADCCEKQEPERNECFLSHKDDSPDLPKLKPDPNTLCDEFKAD EKKFWGKYLYEIARRHPYFYAPELLYYANKYNGVFQECCQAEDKGACLLPKIETMREKVLTSSAR QRLRCASIQKFGERALKAWSVARLSQKFPKAEFVEVTKLVTDLTKVHKECCHGDLLECADDRAD LAKYICDNQDTISSKLKECCDKPLLEKSHCIAEVEKDAIPENLPPLTADFAEDKDVCKNYQEAKD AFLGSFLYEYSRRHPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQ NCDQFEKLGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGKVGTRCCTKPESERMPCTEDYLSL ILNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADICTLPDTEK QIKKQTALVELLKHKPKATEEQLKTVMENFVAFVDKCCAADDKEACFAVEGPKLVVSTQTALA

[0098] Preferably, the albumin has at least 70% sequence identity to HSA (SEQ ID NO: 1), more preferably from 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.2, 99.4, 99.6, or 99.8 to 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.2, 99.4, 99.6, 99.8 or 100% identity to HSA (SEQ ID NO: 1). For example, a preferred albumin has, at most, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutations relative to wild-type HSA (SEQ ID NO: 1).

[0099] Preferably, the albumin comprises at least 175 contiguous amino acids from an albumin having at least 70% sequence identity to HSA (SEQ ID NO: 1), such as from 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550 or 575 to 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575 or 580 amino acids. A fragment may comprise, consist of or substantially correspond to one or more (e.g., several) domains of albumin such as HSA (SEQ ID NO: 1), or variant thereof, such as amino acids corresponding to Domain I (residues 1 to 194 ± 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids), Domain II (residues 192 to 387 ± 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids) or Domain III (residues 381 to 585 ± 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids).

[0100] Typically, the variant albumin maintains at least one of the major properties of the parent albumin or a similar tertiary structure as HSA. For the purposes of the present invention, the sequence identity between two amino acid sequences may be determined using the Needleman-Wunsch algorithm (Needleman & Wunsch, 1970, J Mol Biol 48(3):443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al, 2000, Trends Genet 16(6):276-277), preferably version 5.0.0 or later. The typical parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of "Needle labelled "identity” may be used as the percent identity, this output is the percentage of identical matches between the two sequences over the reported aligned region (including any gaps in the length). Alternatively, the output of Needle labelled "longest identity" (obtained using the -nobrief option) may be used as the percent identity and may be calculated as follows: (Identical Residues x 100) / (Length of Alignment- Total Number of Gaps in Alignment).

[0101] Examples of albumin variants include those described in WO 2011 / 051489, WO 2011 / 124718, WO 2012 / 059486, WO 2012 / 150319, WO 2014 / 072481, WO

[0102] 2013 / 135896, WO 2015 / 036579, WO 2010 / 092135, WO 2013 / 075066, WO

[0103] 2014 / 179657, WO 2009 / 126920, WO 2010 / 059315, WO 2011 / 103076, WO

[0104] 2012 / 112188, WO 2015 / 063611, and WO 2017 / 029407 (the contents of which are incorporated herein by reference in their entirety, and in particular references to albumin variants).

[0105] The term 'parent' or 'parent albumin' as described herein includes the meaning of a human or other animal (e.g., mammalian) albumin. Preferably, the other mammalian albumin is an albumin from a clinically relevant animal such as a mouse, rat, rabbit, dog or guinea pig. The parent may be a naturally occurring (wild-type) polypeptide or an allele thereof or a variant as described above.

[0106] The term "wild-type albumin" as described herein includes the meaning of an albumin having the same amino acid sequence as the predominant allelic variant of albumins naturally found in an animal or in a human being. SEQ ID NO: 1 is an example of a wild-type albumin, being wild-type albumin from Homo sapiens.

[0107] In one embodiment, the albumin is ovalbumin. Ovalbumin (OVA) or albumen refers to the main protein found in egg white. In one embodiment, the egg is an avian egg, such as a chicken egg. In one embodiment, the ovalbumin is chicken ovalbumin (UniProtKB / Swiss-Prot: P01012.2), according to SEQ ID NO: 5:

[0108] MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPG FGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLE PINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQ AMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESII NFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKIS QAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSP

[0109] In one embodiment, the ovalbumin is a variant of a wild-type ovalbumin, optionally wherein the variant ovalbumin has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the wild-type ovalbumin, such as at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with wild-type chicken ovalbumin according to SEQ ID NO: 5.

[0110] In one embodiment, the ovalbumin has at least 70% sequence identity to wild-type chicken ovalbumin (SEQ ID NO: 5), more preferably from 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.2, 99.4, 99.6, or 99.8 to 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.2, 99.4, 99.6, 99.8 or 100% identity to wild-type chicken ovalbumin (SEQ ID NO: 5). For example, the ovalbumin may have 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid mutations relative to wild-type chicken ovalbumin (SEQ ID NO: 5).

[0111] Preferably, the ovalbumin has 100% identity to wild-type chicken ovalbumin (SEQ ID NO: 5).

[0112] In one embodiment, the albumin is recombinant albumin. In another embodiment, the albumin is a serum albumin. In one embodiment, the albumin is recombinant human albumin or human serum albumin. The recombinant human albumin and the human serum albumin may have similar abilities at enhancing transduction. The recombinant ovalbumin and the egg-derived ovalbumin may have similar abilities at enhancing transduction.

[0113] In one embodiment, the albumin is sourced from a recombinant source or is sourced from serum. By "sourced from a recombinant source", we include the meaning that the albumin may be sourced from a recombinant organism such as a recombinant microorganism, recombinant plant or recombinant animal.

[0114] Since some users prefer animal-free ingredients, it is more preferred that the albumin is sourced from a non-animal recombinant source, such as a recombinant microorganism or recombinant plant. Preferred organisms include prokaryotes and, more preferably, eukaryotes such as animals, plants, fungi or yeasts, for example, but not limited to, the following species in which albumins have been successfully expressed as recombinant proteins, for example see the following citations which are incorporated herein by reference: fungi (including but not limited to Aspergillus (WO06066595), Kluyveromyces (Fleer 1991, Bio / technology 9, 968-975), Pichia (Kobayashi 1998 Therapeutic Apheresis 2, 257-262) and Saccharomyces (Sleep 1990, Bio / technology 8, 42-46)) animals (Barash 1993, Transgenic Research 2, 266-276) plants (including but not limited to potato and tobacco (Sijmons 1990, Biotechnology 8, 217 and Farran 2002, Transgenic Research 11, 337-346) and rice e.g., Oryza sativa) mammalian cells such as CHO and HEK prokaryotes (Pandjaitab 2000, J. Allergy Clin. Immunol. 105, 279-285)) e.g., E. coll (EP73646).

[0115] In one embodiment, the albumin is: a) yeast-derived albumin, optionally wherein the yeast Pichia such as Pichia pastoris, Saccharomyces such as Saccharomyces cerevisiae, Candida, or Kluyveromyces such as Kluyveromyces lactis or Kluyveromyces marxianus, Hansenula polymorpha, Schizosaccharomyces pombe, Yarrowia lipolytica, Arxula adeninivorans, Candida such as Candida utilis or Zygosaccharomyces such as Zygosaccharomyces bailii; or b) a plant-derived albumin, optionally a rice-derived albumin. Preferably, the albumin is derived from Saccharomyces, most preferably Saccharomyces cerevisiae (Celik and Calik (Biotechnology Advances, 2012, 30(5): 1108-1118), Gunduz Ergun et al, "Established and Upcoming Yeast Expression Systems" in "Recombinant Protein Production in Yeast", 2019, volume 1923, pages 1- 74. Editors: Gasser and Mattanovich, Publisher: Humana New York, NY.). Particularly preferred examples of albumins (e.g., recombinant human albumin) include those manufactured in yeast, particularly in Saccharomyces cerevisiae, such as the following known commercial presentations of recombinant yeast-derived albumin: Recombumin® Prime (formerly Recombumin®), Recombumin® Elite (formerly AlbIX®), Recombumin® Alpha (formerly Albucult® (all sourced from Albumedix Ltd), or any preparation that is similar thereto.

[0116] In a preferred embodiment, the pre-mix and / or the albumin composition is substantially free or free of other components such as any one or more of an antibody, a peptide, a protein, a lipid, a carbohydrate, a small molecule or an active pharmaceutical ingredient (API) or biopharmaceutical (e.g., a biopharmaceutical that has been classified as Class I, II, III or IV according to the Biopharmaceutics Classification Scheme (BCS) that was first proposed by Amidon et al (Pharm Res. 1995, 12(3):413-420)). In other words, the pre-mix preferably comprises albumin and the retroviridae, and optionally one or more of a buffer and an excipient of an albumin formulation as described herein.

[0117] In one embodiment, the albumin in the pre-mix is present at a concentration of from about 1 milligram / millilitre (mg / mL) to about 400 mg / mL, such as from about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4,

[0118] 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 235, 240, 245, 250 mg / mL to about 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 355, 360, 365, 370, 375, 380, 385, 390,

[0119] 392.5, 395, 397.5, 398, 399, 400 mg / mL. Preferably, the albumin in the pre-mix has a concentration of from about 2 to about 400 mg / mL such as from about 2.5 to about 400 mg / mL, from about 3 to about 400 mg / mL, from about 3.5 to about 400 mg / mL, from about 4 to about 400 mg / mL, from about 4.5 to about 400 mg / mL, from about 4.75 to about 400 mg / mL, from about 10 to about 250 mg / mL, from about 25 to about 250 mg / mL, from about 95 to about 210 mg / mL, from about 10 to about 100 more preferably from about 25 to about 100 mg / mL, and most preferably about 50 mg / mL.

[0120] Typically, the pre-mix is contacted with the cells that are located in a receptacle or vessel such as a well of a plate such as a multi-well plate. It will be appreciated that once the pre-mix is added to the cells, the albumin may be diluted, and so the albumin concentration in the composition comprising the pre-mix and the cells (e.g., the composition in the well of a multi-well plate) may be lower. For example, the albumin concentration in the composition comprising the pre-mix and the cells (e.g., the composition in the well of a multi-well plate) may be diluted by up to about 100-fold compared to the albumin concentration in the pre-mix prior to its addition to the cells. Thus, in one embodiment, the composition comprising the pre-mix and cells (e.g., the composition in the well of a multi-well plate) contains albumin at a concentration of from about 0.01 mg / mL to about 4 mg / mL, such as from about 0.01, 0.025, 0.05, 0.075, 0.100, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6 or 0.65 to about 0.02, 0.025, 0.05, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.25, 0.5, 0.75, 0.8, 0.85, 0.9, 0.95, 0.975,1, 1.5, 2, 2.5, 3, 3.5, 3.6, 3.7, 3.8, 3.9 or 4 mg / mL. Preferably, the albumin in the composition comprising the pre-mix and the cells (e.g., the composition in the well of a multi-well plate) is at a concentration of from about 0.02 to about 4 mg / mL, about 0.025 to about 4 mg / mL, about 0.03 to about 4 mg / mL, about 0.035 to about 4 mg / mL, about 0.04 to about 4 mg / mL, about 0.045 to about 4 mg / mL, about 0.0475 to about 4 mg / mL, about 0.1 to about 2.5 mg / mL, about 0.25 to about 2.5 mg / mL, about 0.95 to about 2.1 mg / mL, about 0.1 to about 1 mg / mL, more preferably about 0.25 to about 1 mg / mL, most preferably about 0.5 mg / mL.

[0121] It will be appreciated that the pre-mix can contact the cell ex vivo or in vitro, or in vivo.

[0122] In one embodiment, the retroviridae is an orthoretrovirinae or spumaretrovirinae, or any virus derived therefrom. Viruses of the subfamily orthoretrovirinae are defined by the ability to reverse transcribe an RIMA genome into DNA that integrates into the cell genome during the intracellular virus life cycle. Viruses of the subfamily spumaretrovirinae contain double-stranded DNA.

[0123] In one embodiment, the orthoretrovirinae is a lentivirus or any virus derived therefrom. In another embodiment, the orthoretrovirinae is any of an alpharetrovirus, a beta retrovirus, a deltaretrovirus, an epsilonretrovirus, a gammaretrovirus, or any virus derived therefrom.

[0124] In one embodiment, the gammaretrovirus is any of a murine leukemia virus, an Abelson murine leukemia virus, a friend virus, a feline leukemia virus, a koala retrovirus (KoRV), a Xenotropic murine leukemia virus-related virus, or any virus derived therefrom.

[0125] In one embodiment, the spumaretrovirinae is any of a bovispumavirus, an equispumavirus, a felispumavirus, a prosimiispumarivirus, or a simiispumavirus, or any virus derived therefrom.

[0126] In one embodiment, the lentivirus is any of a human immunodeficiency virus (HIV) such as HIV-1 or HIV-2, a simian immunodeficiency virus (SIV), a murine lentivirus, an equine infectious encephalitis virus , an equine infectious anemia virus (EIAV), a caprine arthritis encephalitis virus (CAEV), a bovine immunodeficiency virus (BIV) or a feline immunodeficiency virus (FIV), or any virus derived therefrom.

[0127] In one embodiment, the retroviridae is any of a viral particle, a viral vector such as a retroviridae viral vector, or a vector virus.

[0128] The term retroviridae as described herein includes a virus, a viral particle, a viral fragment, a virus-like particle (VLP), a viral vector or a vector virus. As used herein, the terms 'viral particle' and 'virus-like particle' refer to a non-replicating, viral shell, derived from any of several viruses discussed above. Viral particles and VLPs are generally composed of one or more viral proteins, such as, but not limited to those proteins referred to as capsid, coat, shell, surface and / or envelope proteins, or particleforming polypeptides derived from these proteins. In addition, they may or may not contain additional genetic material. The term 'virus particle' refers to the whole virus being infectious unless attenuated or inactivated. VLPs are non-infectious particles. As used herein, the term 'viral fragment' refers to a part or portion of the virus or viral particle, that is preferably non-replicating. As used herein, the terms 'viral vector' and 'vector virus' refer to carriers (e.g., modified viruses) used to deliver genetic material into cells. Viral vectors use the mechanisms that viruses have evolved to efficiently transport their genomes into cells they infect. Viral vectors and vector viruses are commonly used in basic research, gene therapy and the development of vaccines. Within the context of this invention, a lentiviral vector means a nonreplicating vector for the transduction of a cell with a transgene comprising c / s-acting lentiviral RIMA or DNA sequences, and requiring lentiviral proteins (e.g., one or more of or all of Gag, Pol, and / or Env) that are provided in trans. The lentiviral vector may lack expression of one or more or all of functional Gag, Pol, and Env proteins. The lentiviral vector may be present in the form of an RNA or DNA molecule, depending on the stage of production or development of said retroviral vectors. For the avoidance of doubt, it will be appreciated that transduction, the process wherein foreign nucleic acid is introduced into another cell via a viral vector, is distinct from transfection, the process of introducing nucleic acids into cells by non-viral methods.

[0129] The lentiviral vector can be in the form of a recombinant DNA molecule, such as a plasmid. The lentiviral vector can be in the form of a lentiviral vector particle such as an RNA molecule(s) within a complex of lentiviral and other proteins. Typically, lentiviral vector particles, which correspond to modified or recombinant lentivirus particles, comprise a genome which is composed of two copies of single-stranded RNA. These RNA sequences can be obtained by transcription from a double-stranded DNA sequence inserted into a cell genome (proviral vector DNA) or can be obtained from the transient expression of plasmid DNA (plasmid vector DNA) in a transformed cell.

[0130] Typically, the lentiviral vector particles have the capacity for integration. As such, they contain a functional integrase protein. Non-integrating vector particles have one or more mutations that eliminate most or all of the integrating capacity of the lentiviral vector particles. For, example, a non-integrating vector particle can contain mutations in the integrase encoded by the lentiviral pol gene that causes a reduction in integrating capacity. In contrast, an integrating vector particle comprises a functional integrase protein that does not contain any mutations that eliminate most or all of the integrating capacity of the lentiviral vector particles.

[0131] Lentiviral vectors derive from lentiviruses, in particular HIV-1 or HIV-2, SIV, EIA V, CAEV, BIV and FIV, which are modified to remove genetic determinants involved in pathogenicity and introduce new determinants useful for obtaining therapeutic effects.

[0132] Such vectors are based on the separation of the cis- and trans-acting sequences. In order to generate replication-defective vectors, the trans-acting sequences (e.g., gag, pol, tat, rev, and env genes) can be deleted and replaced by an expression cassette encoding a transgene. In one embodiment, the retroviridae is a live retroviridae, an attenuated retroviridae, a live-attenuated retroviridae.

[0133] In one embodiment, the retroviridae is in the form of a vaccine, preferably wherein the vaccine is a viral-based vaccine. Lentiviral vectors have a high potential for transducing dendritic cells in vivo. Within these cells, which are the most efficient at activating naive T cells, lentiviral vectors induce endogenous expression of transgenic antigens that directly access antigen presentation pathways without the need for external antigen capture or cross-presentation. For, example the retroviridae-based vaccine may protect against (i) viruses, such as viruses from the flaviridae family (such as West Nile virus (WNV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), Dengue virus (DENV)), SARS-CoV-2; bacterial infections (such as tuberculosis) or parasitic infection (such as malaria).

[0134] In one embodiment, the retroviridae in the pre-mix is present at a multiplicity of infection range of 0.1-100, such as from about 0.1, 0.25, 0.5, 0.75, 1, 2, 5, 6, 7, 8, 10, 15, 25, 30, 35, 40, 45, 50, 55 to about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100. Preferably, the retroviridae in the pre-mix is present at an MOI range of 1-10. Most preferably, the retroviridae in the pre-mix is present at an MOI of 5.

[0135] By "multiplicity of infection" or "MOI", we include the meaning of the ratio of number of virus particles to number of target cells present. In general, as the MOI increases, the percentage of cells infected with at least one viral particle also increases. Those skilled in the art would be capable of determining MOI, as number of infectious virus particles

[0136] - - - - - - . The optimum MOI can be determined number of target cells experimentally by those skilled in the art.

[0137] In one embodiment, the pre-mix further comprises a buffer. For example, one or both of the retroviridae and the albumin may be provided in a buffer, and mixed together to form a pre-mix that comprises a buffer. Alternatively, the retroviridae and albumin may both be added to a buffer to a form a pre-mix that comprises the retroviridae, albumin and a buffer.

[0138] In one embodiment, the buffer has a buffering capacity from about pH 6.0 to about pH 8.0, such as from about pH 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 to about pH 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0. In one embodiment, the buffer is any of phosphate-buffered saline (PBS), Hanks buffered salt solution, Earle's balanced salt solution, Tyrode's solution, MOPS buffer, HEPES buffer or any isotonic solutions used for injection, such as Plasma-Lyte®, e.g., Plasma-Lyte® 148, or Ringer's lactate. Preferably, the buffer is PBS.

[0139] In one embodiment, the pre-mix comprises 1-250 millimolar (mM) of buffer. The buffer concentration may be from about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mM to about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200 or 250 mM. A buffer concentration from about 10 mM to about 200 mM is preferred, particularly about 150 mM, particularly wherein the buffer is PBS.

[0140] The albumin that is added to the retroviridae to form the pre-mix may be provided in the form of an albumin formulation. The formulation is typically a liquid albumin formulation although it is appreciated that the formulation may be a dried formulation such as a freeze-dried formulation.

[0141] In one embodiment, the albumin which is used to form the pre-mix is provided in the form of a formulation, and suitable formulations are any of the group consisting of: (a) from 13 to 16 mM, preferably about 14.5 mM, of sodium, from 2.9 to 3.5 mM, preferably about 3.2 mM, of octanoate, from 1 to 2 mg / L, preferably from 1 to 1.5 mg / L, of Polysorbate 80, from 19 to 21 mg / mL, preferably about 20 mg / mL, of albumin (weight / volume (w / v)), wherein the pH is 6.7 to 7.3, preferably about 7; (b) from 12 to 16 mM, preferably about 14.5 mM, of sodium, from 0.4 to 1.2 mM, preferably about 0.8 mM, of octanoate, from 0 to 5 mg / L, preferably about 2.5 to 4.5 mg / L, of Polysorbate 80, from 9.5 to 10.5 mg / mL, preferably about 10 mg / mL, of albumin (w / v), wherein the pH is 6.4 to 7.4, preferably about 7; (c) from 12 to 16 mM, preferably about 14.5 mM, of sodium, from 0.8 to 2.4 mM, preferably about 1.6 mM, of octanoate, from 0 to 10 mg / L, preferably from 5 to 7 mg / L, of Polysorbate 80, from 19 to 21 mg / mL, preferably about 20 mg / mL, of albumin (w / v), wherein the pH is 6.4 to 7.4, preferably about 7; (d) from 20 to 30 mM, preferably from 23 to 26 mM, of sodium, from 0 to 0.3 mM, preferably from 0 to 0.1 mM of octanoate, from 9.5 to 10.5 mg / mL, preferably about 10 mg / mL, of albumin (w / v), wherein the pH is from 6 to 7, preferably about 6.5.

[0142] In another embodiment, suitable formulations are any of the group consisting of: (a) from 130 to 160 mM, preferably about 145 mM, of sodium, from 29 to 35 mM, preferably about 32 mM, of octanoate, from 10 to 20 mg / L, preferably from 10 to 15 mg / L, of Polysorbate 80, from 190 to 210 mg / mL, preferably about 200 mg / mL, of albumin (weight / volume (w / v)), wherein the pH is 6.7 to 7.3, preferably about 7; (b) from 120 to 160 mM, preferably about 145 mM, of sodium, from 4 to 12 mM, preferably about 8 mM, of octanoate, from 0 to 50 mg / L, preferably from 25 to 45 mg / L, of Polysorbate 80, from 95 to 105 mg / mL, preferably about 100 mg / mL, of albumin (w / v), wherein the pH is 6.4 to 7.4, preferably about 7; (c) from 120 to 160 mM, preferably about 145 mM, of sodium, from 8 to 24 mM, preferably about 16 mM, of octanoate, from 0 to 100 mg / L, preferably from 50 to 70 mg / L, of Polysorbate 80, from 190 to 210 mg / mL, preferably about 200 mg / mL, of albumin (w / v), wherein the pH is 6.4 to 7.4, preferably about 7; (d) from 200 to 300 mM, preferably from 230 to 260 mM, of sodium, from 0 to 3 mM, preferably from 0 to 1 mM of octanoate, from 95 to 105 mg / mL, preferably about 100 mg / mL, of albumin (w / v), wherein the pH is from 6 to 7, preferably about 6.5.

[0143] In a further embodiment, suitable formulations are any of the group consisting of (a) from 6.5 to 8 mM, preferably about 7.25 mM, of sodium, from 1.45 to 1.75 mM, preferably about 1.6 mM, of octanoate, from 0.5 to 1 mg / L, preferably from 0.5 to 0.75 mg / L, of Polysorbate 80, from 9.5 to 10.5 mg / mL, preferably about 10 mg / mL, of albumin (weight / volume (w / v)), wherein the pH is 6.7 to 7.3, preferably about 7; (b) from 6 to 8 mM, preferably about 7.25 mM, of sodium, from 0.2 to 0.6 mM, preferably about 0.4 mM, of octanoate, from 0 to 2.5 mg / L, preferably from 1.25 to 2.25 mg / L, of Polysorbate 80, from 4.75 to 5.25 mg / mL, preferably about 5 mg / mL, of albumin (w / v), wherein the pH is 6.4 to 7.4, preferably about 7; (c) from 6 to 8 mM, preferably about 7.25 mM, of sodium, from 0.4 to 1.2 mM, preferably about 0.8 mM, of octanoate, from 0 to 5 mg / L, preferably about 2.5 to 3.5 mg / L, of Polysorbate 80, from 9.5 to 10.5 mg / mL, preferably about 10 mg / mL, of albumin (w / v), wherein the pH is 6.4 to 7.4, preferably 7; (d) from 10 to 15 mM, preferably from 11.5 to 13 mM, of sodium, from 0 to 0.15 mM, preferably from 0 to 0.05 mM of octanoate, from 4.75 to 5.25 mg / mL, preferably about 5 mg / mL, of albumin (w / v), wherein the pH is from 6 to 7, preferably about 6.5.

[0144] In one embodiment, the albumin formulation contains albumin at a concentration from 2 to 400 mg / mL such as from 2.5 to 400 mg / mL, from 4.5 to 400 mg / mL, or from 4.75 to 400 mg / mL, preferably from 10 to 250 mg / mL mM, such as from 95 to 210 mg / mL, more preferably from 10 to 100 mg / mL, most preferably from about 10 to about 20 mg / mL. In one embodiment, the albumin formulation contains sodium at a concentration from 0.4 to 300 mM or 1 to 300 mM (e.g., from 50-300 mM or 120-300 mM), preferably from 4 to 25 mM or 10 to 50 mM, more preferably from 5 to 14 mM or 12 to 30 mM, most preferably about 11 mM or about 25 mM.

[0145] In one embodiment, the albumin formulation contains octanoate at a concentration from 0 to 35 mM or 0 to 16 mM, preferably from 0 to 5 mM or 0 to 2.5 mM, more preferably from 0 to 3.5 mM or 0 to 1.6 mM, most preferably about 0.1 mM or about 0.04 mM.

[0146] In one embodiment, the albumin formulation contains Polysorbate 80 at a concentration from 0 to 100 mg / mL or 0 to 45 mg / mL, preferably from 0 to 10 mg / mL or 0 to 4.5 mg / mL, more preferably from 0 to 5 mg / mL or 0 to 2.5 mg / mL, most preferably about 2.5 mg / mL or about 1.1 mg / mL.

[0147] In one embodiment, the pre-mix comprises from 2 to 400 mg / mL or 2.5 to 400 mg / mL of albumin (w / v) such as from 4.5 to 400 mg / mL or 4.75 to 400 mg / mL of albumin (w / v), preferably from 10 to 250 mg / mL, such as from 95 to 210 mg / mL, more preferably from 10 to 100 mg / mL, most preferably from about 10 to about 20 mg / mL of albumin (w / v).

[0148] In one embodiment, the pre-mix comprises from 0.4 to 300 mM or 1 to 300 mM, preferably from 4 to 50 mM or 10 to 50 mM, more preferably from 5 to 30 mM or 12 to 30 mM, most preferably about 11 mM or about 25 mM of sodium.

[0149] In another embodiment, the pre-mix comprises 0.7 to 270 mM (e.g., from 84-270 mM), preferably from 7 to 45 mM, more preferably from 8.4 to 27 mM, most preferably about 22.5 or about 17.5 mM of sodium.

[0150] In one embodiment, the pre-mix comprises from 0 to 35 mM or 0 to 16 mM, preferably from 0 to 5 mM or 0 to 2.5 mM, more preferably from 0 to 3.5 mM or 0 to 1.6 mM, most preferably about 0.1 mM or about 0.04 mM of octanoate.

[0151] In another embodiment, the pre-mix comprises from 0 to 24.5 mM or 0 to 31.5 mM, preferably from 0 to 3.5 mM or 0 to 4.5 mM, more preferably from 0 to 2.5 mM or 0 to 3.2 mM, most preferably about 0.07 or about 0.09 mM of octanoate. In one embodiment, the pre-mix comprises from 0 to 100 mg / mL or 0 to 45 mg / mL, preferably from 0 to 10 mg / mL or 0 to 4.5 mg / mL, more preferably from 0 to 5 mg / mL or 0 to 0.25 mg / mL, most preferably about 2.5 mg / mL or about 1.1 mg / mL of Polysorbate 30 or Polysorbate 80, preferably Polysorbate 80.

[0152] In another embodiment, the pre-mix comprises from 0 to 70 mg / mL or 0 to 90 mg / mL, preferably from 0 to 7 mg / mL or 0 to 9 mg / mL, more preferably from about 0 to 3.5 mg / mL or about 0 to 4.5 mg / mL, most preferably about 1.75 mg / mL or about 2.25 mg / mL of Polysorbate 30 or Polysorbate 80, preferably Polysorbate 80.

[0153] In one embodiment, the pre-mix comprises from 2 to 400 mg / mL or 2.5 to 400 mg / mL of albumin (w / v) such as from 4.5 to 400 mg / mL or 4.75 to 400 mg / mL of albumin (w / v) in combination with from 0.4 to 300 or 1 to 270 or 1 to 300 mM of sodium.

[0154] In one embodiment, the pre-mix comprises from 2 to 400 mg / mL or 2.5 to 400 mg / mL of albumin (w / v) such as from 4.5 to 400 mg / mL or 4.75 to 400 mg / mL of albumin (w / v) in combination with from 0 to 35 mM or 0 to 16 or 0 to 31.5 mM of octanoate.

[0155] In one embodiment, the pre-mix comprises from 2 to 400 mg / mL or 2.5 to 400 mg / mL of albumin (w / v) such as from 4.5 to 400 mg / mL or 4.75 to 400 mg / mL of albumin (w / v) in combination with from 0 to 100 mg / mL or 0 to 45 or 0 to 90 mg / mL of Polysorbate 30 or Polysorbate 80, preferably Polysorbate 80.

[0156] In one embodiment, the pre-mix comprises from 2 to 400 mg / mL or 2.5 to 400 mg / mL of albumin (w / v) such as from 4.5 to 400 mg / mL or 4.75 to 400 mg / mL of albumin (w / v) in combination with from 0.4 to 300 mM, 1 to 270 mM or 1 to 300 mM of sodium and from 0 to 16 mM, 0 to 31.5 mM or 0 to 35 mM of octanoate.

[0157] In one embodiment, the pre-mix comprises from 2 to 400 mg / mL or 2.5 to 400 mg / mL of albumin (w / v) such as from 4.5 to 400 mg / mL or 4.75 to 400 mg / mL of albumin (w / v) in combination with from 0.4 to 300 mM, 1 to 270 mM or 1 to 300 mM of sodium and from 0 to 45 mg / mL, 0 to 90 mg / mL or 0 to 100 mg / mL of Polysorbate 30 or Polysorbate 80, preferably Polysorbate 80.

[0158] In one embodiment, the pre-mix comprises from 2 to 400 mg / mL or 2.5 to 400 mg / mL of albumin (w / v) such as from 4.5 to 400 mg / mL or 4.75 to 400 mg / mL of albumin (w / v) in combination with from 0 to 16 mM, 0 to 31.5 mM or 0 to 35 mM of octanoate and from 0 to 45 mg / mL, 0 to 90 mg / mL or 0 to 100 mg / mL of Polysorbate 30 or Polysorbate 80, preferably Polysorbate 80.

[0159] In one embodiment, the pre-mix and / or the albumin composition is substantially free of or free of fatty acids.

[0160] In one embodiment, the enhancement of transduction is greater than or equal to the enhancement of transduction provided for by using a known transduction enhancer (e.g., replacing albumin in the pre-mix with a known transduction enhancer, or adding a known transduction enhancer to the cells without forming a pre-mix). Transduction enhancers are known in the art and include enhancers such as Lentiboost® (Sirion Biotech), Polybrene (Merck Life Sciences), Poloxamer 338 (also known as Poloxamer Synperonic® F108), Lenti-X™ Accelerator (Takara Bio Inc), RetroNectin® (Takara Bio Inc), Vectofusion® (Miltenyi Biotec Ltd) or protamine sulphate or a combination thereof. Thus is one embodiment, the enhancement of transduction is greater than or equal to the enhancement of transduction provided for by any one or more of Lentiboost® (Sirion Biotech), Polybrene (Merck Life Sciences), Poloxamer 338 (also known as Poloxamer Synperonic® F108), Lenti-X™ Accelerator (Takara Bio Inc), RetroNectin® (Takara Bio Inc), Vectofusion® (Miltenyi Biotec Ltd) or protamine sulphate or a combination thereof.

[0161] By the enhancement of transduction is greater than the enhancement of transduction provided for by using a known transduction enhancer, we include the meaning that the transduction is increased by at least 1% compared to the transduction provided for by using a known transduction enhancer, more preferably the transduction is increased by at least 2%, 3%, 4% or 5%, such as at least 7.5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%. It is preferred that the transduction is increased by at least 5% compared to the transduction provided for by using a known transduction enhancer.

[0162] In one embodiment, the cell is further contacted with one or more known transduction enhancers, such as any one or more of Lentiboost® (Sirion Biotech), Polybrene (Merck Life Sciences), Poloxamer 338 (also known as Poloxamer Synperonic® F108), Lenti-X™ Accelerator (Takara Bio Inc), RetroNectin® (Takara Bio Inc), Vectofusion® (Miltenyi Biotec Ltd or protamine sulphate or a combination thereof. Preferably, the one or more transduction enhancers are added to the cells before the pre-mix is added to the cells. However, it will be appreciated that the one or more transduction enhancers may form part of the pre-mix that, once formed, is added to the cells.

[0163] It is appreciated that when the cell is further contacted with one or more known transduction enhancers, the enhancement of transduction may be greater than the transduction provided for when the cell is contacted with only the pre-mix without one or more known transduction enhancers and / or when the cell is contacted with only the one or more known transduction enhancers without the pre-mix. The transduction may be increased by at least 1% compared to the transduction provided for when the cell is contacted with only the pre-mix without one or more known transduction enhancers and / or when the cell is contacted with only the one or more known transduction enhancers without the pre-mix, more preferably by at least 2%, 3%, 4%, or 5%, such as at least 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70% or 75%, 100%, 150% or 200%. It is preferred that the transduction is increased by at least 5% compared to the transduction provided for when the cell is contacted with only the pre-mix without one or more known transduction enhancers and / or when the cell is contacted with only the one or more known transduction enhancers without the pre-mix. For example, it is preferred that the transduction is increased by at least 5% compared to the transduction provided for by when the cell is contacted with only the pre-mix without one or more known transduction enhancers and by at least 5% when the cell is contacted with only the one or more known transduction enhancers without the pre-mix. Most preferably, the enhancement of transduction when the cell is contacted with the pre-mix and with one or more known transduction enhancers is greater than the additive enhancement of transduction attributable, individually, to the pre-mix and to the one or more known transduction enhancers. In other words, the addition of the pre-mix and the one or more known transduction enhancers may result in a synergistic effect on the enhancement of transduction.

[0164] Lentiboost® is a commercial transduction enhancer and has a proprietary formulation comprising Poloxamer 338 and optionally Polybrene.

[0165] In one embodiment, the concentration of Lentiboost® in the composition comprising the pre-mix and cells is about 0.01 mg / mL to about 10 mg / mL, such as from about 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2, 3, 4, 5 or 6 to about 0.05, 0.1, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 9.5, 9.8, 9.9 or 10 mg / mL. Preferably, the concentration of Lentiboost® in the composition comprising the pre-mix and cells is about 0.1 to about 2 mg / mL. Most preferably, the concentration of Lentiboost® in the composition comprising the pre-mix and cells is 1 mg / mL.

[0166] In one embodiment, the concentration of Lentiboost® in the pre-mix is about 0.1 mg / mL to about 1000 mg / mL, such as from about 0.1, 0.25, 0.5, 0.75, 1, 5, 10, 50, 100, 150, 200, 300, 400, 500 or 600 to about 200, 300, 400, 500, 600, 700, 800, 900, 950, 980, 990 or 1000 mg / mL. Preferably, the concentration of Lentiboost® in the pre-mix is about 10 to about 200 mg / mL, such as from about 10 to about 100 mg / mL. Most preferably, the concentration of Lentiboost® in the pre-mix is 0.1 - 5 mg / mL, such as about 0.2, 0.3 or 0.4 mg / mL.

[0167] Poloxamer 388 or Poloxamer Synperonic® F108 is a 'large non-ionic amphiphilic molecule', as defined in WO 2013 / 127964, according to the following formula:

[0168] In one embodiment, the concentration of Poloxamer 338 or Poloxamer Synperonic® F108 in the composition comprising the pre-mix and cells (e.g., in a well of a multiwell plate) is about 0.5 pg / mL to about 50 pg / mL, such as from about 0.5, 0.55, 0.6, 0.65, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 pg / mL to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 46, 47, 48, 49 or 50 pg / mL.

[0169] In one embodiment, the concentration of Poloxamer 338 or Poloxamer Synperonic® F108 in the pre-mix is from about 50 pg / mL to about 5000 pg / mL, such as from about 50, 55, 60, 65, 70, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 pg / mL to about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 4600, 4700, 4800, 4900 or 5000 pg / mL.

[0170] Polybrene is a polycation l,5-dimethyl-l,5-diaza-undeca-methyl-polymethobromide, as defined in WO 2013 / 127964, according to the following formula:

[0171] Polybrene reduces charge repulsion between the virus and the cellular membrane. The optimum final concentration of Polybrene may be determined by a person skilled in the art. Excessive exposure (such as more than 24 hours) to Polybrene can be toxic to cells.

[0172] In one embodiment, the concentration of Polybrene in the composition comprising the pre-mix and cells (e.g., in a well of a multi-well plate) is about 1 to about 20 pg / mL, such as from about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0. 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13 or 14 to about 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 19.5 or 20 pg / mL. Preferably, the concentration of Polybrene in the composition comprising the pre-mix and cells is from about 2 to about 12 pg / mL. Most preferably, the concentration of Polybrene in the composition comprising the pre-mix and cells is about 8 pg / mL.

[0173] In one embodiment, the concentration of Polybrene in the pre-mix is from about 100 pg / mL to about 2 mg / mL, such as from about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 pg / mL, 1, 1.1, 1.2, 1.3 or 1.4 mg / mL to about 900, 950 pg / mL, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.95 or 2 mg / mL. Preferably, the concentration of Polybrene in the pre-mix is from about 200 pg / mL to about 1.2 mg / mL. Most preferably, the concentration of Polybrene in the pre-mix is about 800 pg / mL.

[0174] When the cell is further contacted with one or more transduction enhancers, the enhancement of transduction may be even greater than that already provided by the albumin in the pre-mix (i.e. without the additional step of further contacting the cell with a transduction enhancer). In one embodiment, the enhancement of transduction is at least 1.01, 1.02, 1.03, 1.04, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,

[0175] 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,

[0176] 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,

[0177] 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or at least 10-fold greater than the enhancement of transduction already provided without further contacting the cell with a transduction enhancer. For example, by an enhancement of "at least 1.1-fold", we mean at least a 10% increase in transduction.

[0178] In one embodiment, using albumin and one or more transduction enhancers can have a synergistic effect on the enhancement of transduction. By "synergistic effect", we include the meaning that the increase in transduction is significantly higher than the combined sum of the albumin and the transduction enhancer.

[0179] As shown by the inventors in Example 1, the effect of the albumin on lentivirus was found to vary depending on how long the pre-mix was incubated for before contacting the cells. For example, if the lentivirus was stored in the albumin before being added to the cells, the albumin both stabilised the virus and enhanced transduction, whereas if the albumin was mixed with the lentivirus and then the pre-mix transferred to the cells immediately, the lentivirus only enhanced transduction. Accordingly, in one embodiment, and especially where the pre-mix is incubated for a period of time before contacting the cells in step (ii), the method of the invention also increases the stability of the retroviridae (e.g., retrovirus or lentivirus).

[0180] Thus, the invention also provides a method of (a) enhancing transduction of a retroviridae into a cell and (b) increasing the stability of the retroviridae, the method comprising:

[0181] (i) combining the retroviridae with albumin to form a pre-mix; and

[0182] (ii) contacting the cell with the pre-mix formed in step (i).

[0183] By "stability" of the retroviridae, we include the meaning that the retroviridae remains intact and functional. The stability of a retroviridae can be assessed using any suitable method known in the art. For example, a low stability can affect the quality and efficacy of the retroviridae. This is characterised by a significant reduction in the titre of the retroviridae over time. Existing technologies for increasing the stability of viruses include keeping the formulations at very low temperatures (e.g., -90°C to -70°C). However, loss of viability of the virus occurs following multiple rounds of freeze-thaw cycles or after very short periods of time unfrozen. This can lead to wasting non- negligible amounts of expensive drug or vaccine formulations.

[0184] By "increasing the stability of the retroviridae", we include the meaning that the virus is increased compared to the stability of the retroviridae in the absence of albumin. In other words, when the stability of the virus is assessed under particular conditions, then contacting the virus with albumin should increase the stability of the virus when assessed under the same particular conditions. The conditions selected may be any suitable "stress condition" which is known to affect the stability of the virus, for example any of freeze-thaw cycles, temperature incubation (e.g., at a set temperature (such as room temperature, 25°C, 35°C, or 45°C) for a set period of time (e.g., at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours or 8 hours, at least 24 hours, at least 36 hours, at least 2, 3, 4, 5 or 6 days, at least 1 or 2 weeks, or at least 1 month or 2 months) or at least 2 months storage or incubation at a temperature of from 2 to 8°C, such as from about 2, 3, 4, 5, 6 or 7 to about 3, 4, 5, 6, 7 or 8°C, for example about 4°C.

[0185] Assessing whether stability of a retroviridae is increased may be carried out using any suitable technique known in the art and as described in Example 1. For example, aggregation of the virus may be assessed for example by measuring the hydrodynamic radius of a formulation containing the virus. Dynamic light scattering (DLS) may be used. Stability may be determined by measuring the ability of the virus to bind to a binding partner (e.g., a monoclonal antibody), or to infect a cell.

[0186] Retroviridae are commonly used in therapy and so enhancing the transduction efficiency of the virus into a cell would be especially useful to improve the efficacy of such therapy. Accordingly, in a second aspect, the invention provides a method of improving the efficacy of a retroviridae based therapy, the method comprising combining the retroviridae with albumin to form a pre-mix; and administering the premix to a subject.

[0187] Preferences for the retroviridae, albumin and pre-mix include those described above in relation to the first aspect of the invention.

[0188] By "retroviridae based therapy", we include the meaning of a therapy that is based on retroviridae to treat and / or prevent a disease or condition that would benefit from the therapy. For example, the retroviridae based therapy may be a gene-based therapy, wherein the retroviridae is used as a vector to transfer a particular gene or part thereof to a cell so as to prevent and / or treat a disease or condition. Examples of retroviridae based gene therapy include using lentiviruses to deliver p-globin, gamma chain (yc), and tumour-specific T-cell receptor (TCR). Other examples include ADA cDNA (e.g. for treatment of ADA-SCID), IL2RG cDNA (e.g. for treatment of SCID), CYBB cDNA (for treatment of XL CGD) and CD4 binding agent. Further examples of retroviridae based gene therapy also include using retroviruses to deliver a drug susceptibility or "suicide" gene, such as herpes simplex thymidine kinase (TK), p53, bcl-xs.

[0189] Such therapies can be used to treat and / or prevent diseases and / or conditions. In one embodiment, the disease or condition is a genetic disease or condition. In another embodiment, the disease or condition is a sporadic disease or condition. Examples of such diseases or conditions include but are not limited to as cancer (such as lymphoma, leukemia or multiple myeloma), lysosomal storage diseases, 0-thalassemia, cerebral adrenoleukodystrophy, sickle cell disease, haemophilia, Fanconi anemia, metachromatic leukodystrophy and adrenoleukodystrophy, Wiskott-Aldrich syndrome, Severe combined immunodeficiency (SCID), X-linked severe combined immunodeficiency (XL-SCID), adenosine deaminase severe combined immunodeficiency (ADA-SCID), X-linked chronic granulomatous disease (XL-CGD), Parkinson's disease, macular degeneration.

[0190] By "gene therapy", we include the meaning of a technique that modifies a subject's genes to treat, cure or prevent a disease or disorder. Gene therapies can work by several mechanisms: e.g., replacing a disease-causing gene with a healthy copy of the gene, inactivating a disease-causing gene that is not functioning properly or introducing a new or modified gene into the body to help treat a disease. The transferred genetic material can change how a single protein or group of proteins is produced by the cell.

[0191] The "retroviridae based therapy" may be an immunotherapy. By "immunotherapy", we include the meaning of the treatment or prevention of a disorder or disease that involves the activation, enhancement, reduction, suppression, or desensitisation of the immune system. The retroviridae based therapy maybe a vaccine. Preferably, the disorder or disease is an autoimmune disorder, an allergy, or a cancer.

[0192] The "retroviridae based therapy" maybe an oncolytic viral therapy. By "oncolytic viral therapy", we include the meaning of a form of immunotherapy that uses competent replicating viruses to infect and destroy cancer cells. Preferably, the competent viruses specifically attack tumour cells but not healthy cells.

[0193] The pre-mix may be administered to a subject using any amount and any route of administration effective for preventing and / or treating the disease and / or condition. By "subject", we include the meaning of any organism to which a pre-mix in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include any living organisms, such as animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). Preferably, the subject is in need of a retroviridae based therapy. By "a subject in need of a retroviridae based therapy", we include the meaning of individuals who would benefit from retroviridae based therapy.

[0194] By "improved efficacy" of a retroviridae based therapy, we include the meaning that the therapy achieves the desired clinical result using a lower dosing regimen of the retroviridae based therapy, thereby reducing unwanted side effects. Those skilled in the art would be capable of selecting an appropriate assay to measure the desired clinical result of the agent.

[0195] In one embodiment, the efficacy is improved due to the enhancement of transduction as a result of combining the retroviridae with albumin to form a pre-mix.

[0196] In one embodiment, the pre-mix is administered to the subject immediately after it was formed.

[0197] In another embodiment, the pre-mix is administered to the subject within a timeframe of from about 30 seconds to about 36 hours, such as from about 30, 35, 40, 45, 50, 55 or 60 seconds to about 1, 2, 4, 6, 8, 12, 18, 24, 28, 32, 33, 34, 35 or 36 hours after the pre-mix was formed.

[0198] In the clinical setting, it may not be practical for the medical staff or other staff to contact the pre-mix with the cells within seconds or minutes. Therefore, pre-mixing of albumin and the retroviridae and storing the pre-mix for a longer period of time (including freezing e.g., at about at from about -15°C to about -90°C, e.g., at from about -15°C to about -25°C , e.g., at from about -70°C to about -90°C, e.g., at about -80°C or about -20°C) could simplify the clinical translation of albumin-enhanced retroviridae transduction.

[0199] In yet another embodiment, the pre-mix is administered to the subject not more than about 30 seconds after the pre-mix was formed, or not more than about 5 minutes after the pre-mix was formed, such as not more than about 10, 20, 30, 40, 50 or 60 seconds, not more than about 1, 2, 3, 4 or 5 minutes. In one embodiment, the pre-mix is stored at a temperature of from about 0 to about 35°C, such as from about 0 to about 5°C or from about 15 to about 35°C, or at room temperature before it is administered to the subject, such as from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20°C to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35°C.

[0200] In one embodiment, the pre-mix is stored at a temperature of: i) from about 15 to about 35°C, or at room temperature, for a period of from about 30 seconds to about 36 hours; or ii) from about 0 to about 5°C for a period of from about 30 seconds to about 72 hours before it is administered to the subject, e.g. from about 30 seconds to 48 hours, from about 30 seconds to about 36 hours, from about 30 seconds to about 24 hours, or from about 30 seconds to about 12 hours before it is administered to the subject. When stored at from about 15 to about 35°C (e.g. room temperature), the period of time may be from about 30, 35, 40, 45, 50, 55 or 60 seconds, 1, 2, 5, 10, 15 minutes to about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 32, 33, 34, 35 or 36 hours. When stored at from about 30 seconds to about 72 hours, the period of time may be from about 30, 35, 40, 45, 50, 55 or 60 seconds, 1, 2, 5, 10, 15, 20, 30, 40 or 50 minutes, 1, 2, 3 or 4 hours to about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 35, 36, 40, 44, 48, 52, 56, 60, 64, 68 or 72 hours.

[0201] Retroviridae are commonly used in therapy and specifically in cell therapy. Accordingly, in a third aspect, the invention provides a method of producing a cell therapy ex vivo, wherein the cell therapy comprises cells transduced by a retroviridae, the method comprising: (i) providing the cells to be transduced by the retroviridae, and (ii) carrying out the method of the first aspect of the invention on the cells ex vivo so as to produce the cell therapy. The invention also includes the cell therapy so produced for use in treating a subject in need thereof as described further below.

[0202] By "cell therapy", we include the meaning of a technique where intact, live cells are transferred into a subject to treat, cure or prevent a disease or disorder. Such cells are typically modified using a viral vector before being administered to the subject. In some embodiments, the cells originate from the subject (autologous cells) or a donor (allogeneic cells).

[0203] The cells used in cell therapy can be classified by their potential to transform into different cell types. The cells to be transduced by the retroviridae may be stem cells (such as pluripotent stem cells, somatic stem cells, adult stem cells, cancer stem cells, or tumor-initiating cells) or non-stem cells. For example, the cells may be any of hematopoietic progenitor cells, endothelial progenitor cells, pericytes, fibroblasts, chondrocytes, keratinocytes, hepatocytes, pancreatic islet cells, monocytes, lymphocytes, granulocytes, T cells, B-cells, dendritic cells, natural killer cells, or macrophages. Suitable examples of cells also include those described above in relation to the first aspect of the invention.

[0204] In one embodiment, gene therapy and cell therapy can be combined to treat a genetic disease. Stem cells are altered by gene therapy in culture (ex vivo). The altered cells are then administered to the subject.

[0205] Chimeric antigen receptors (CARs) engineered from three distinct domains (antigen recognition, co-stimulatory signaling, and T-cell signaling) can be introduced into T cells using a lentiviral vector. The cells expressing the modified receptor recognise the antigen of interest and harness the potent cytotoxic activity of T cells to attack tumour cells. In one embodiment, the CAR T-cell therapy targets the CD19 antigen, a protein expressed on B cells and B-cell malignancies.

[0206] A fourth aspect of the invention provides a retroviridae based cell therapy for use in treating a subject, wherein the cell therapy is produced according to the third aspect of the invention.

[0207] It will also be appreciated that the invention includes a method of treating a subject with a cell therapy wherein the cell therapy comprises cells transduced by a retroviridae, the method comprising (i) providing the cells to be transduced by the retroviridae, (ii) carrying out the method of the first aspect of the invention on the cells ex vivo so as to produce the cell therapy, and (iii) administering the cell therapy to the subject in need thereof.

[0208] Preferences for the subject include those described above in relation to the second aspect of the invention. It will be appreciated that the cell therapy in the context of the third aspect of the invention may be considered to be a type of retroviridae based therapy as described above wherein the retroviridae based therapy comprises cells transduced by a retroviridae.

[0209] Preferences for the timing of forming the pre-mix and adding it to the cells include those described above in relation to the first aspect of the invention. A fifth aspect of the invention provides a retroviridae based therapy for use in treating a subject, wherein the retroviridae is combined with albumin to form a pre-mix, which is subsequently administered to the subject.

[0210] It will also be appreciated that the invention includes a method of treating a subject with the retroviridae based therapy, the method comprising (a) forming the pre-mix; and (b) administering the pre-mix to the subject.

[0211] By "treatment" or "treating", we include the meaning of obtaining a therapeutically or prophylactically beneficial or desired result including and preferably a beneficial or desired clinical result. Such beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) cancerous cells or other diseased tissue, decreasing symptoms resulting from a disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and / or prolonging survival of individuals.

[0212] Preferences for the subject include those described above in relation to the second aspect of the invention.

[0213] Preferences for the timing of forming the pre-mix and for administering to a subject include those described above in relation to the first aspect of the invention.

[0214] In one embodiment of the second or fifth aspects, the retroviridae based therapy is an immunotherapy such as oncolytic viral therapy, or a gene therapy, or a vaccine.

[0215] A sixth aspect of the invention provides a use of albumin to form a pre-mix for enhancing transduction of a retroviridae into a cell, wherein the albumin is combined with the retroviridae to form the pre-mix before contacting the cell. In one embodiment, the cell is contacted in vivo. In another embodiment, the cell is contacted ex vivo, such as in vitro.

[0216] A seventh aspect of the invention provides a use of a pre-mix for enhancing transduction of a retroviridae into a cell, wherein the pre-mix comprises albumin and the retroviridae and is formed before contacting the cell. In one embodiment, the cell is contacted in vivo. In another embodiment, the cell is contacted ex vivo, such as in vitro. In one embodiment of the sixth or seventh aspects of the invention, the pre-mix is formed immediately before contacting the cell.

[0217] In one embodiment of the sixth or seventh aspects of the invention, the pre-mix is formed within a timeframe of from about 30 seconds to about 36 hours before contacting the cell, such as from about 30, 35, 40, 45, 50, 55 or 60 seconds to about 1, 2, 4, 6, 8, 12, 18,24, 28, 32, 33, 34, 35 or 36 hours.

[0218] In another embodiment of the sixth or seventh aspects of the invention, the pre-mix is formed not more than about 30 seconds before contacting the cell, or not more than about 5 minutes before contacting the cell, such as not more than about 10, 20, 30, 40, 50 or 60 seconds, not more than about 1, 2, 3, 4 or 5 minutes.

[0219] In yet another embodiment of the sixth or seventh aspects of the invention, the premix is stored at a temperature of: (i) from about 0 to 35°C, or at room temperature, such as from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20°C to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35°C for a period from about 30 seconds to about 72 hours; or (ii) from about 15 to 35°C, or at room temperature, such as from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 °C to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35°C for a period of from about 30 seconds to about 36 hours; or (iii) from about 0 to about 5°C, such as from 0, 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0°C to 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5°C for a period of from about 30 seconds to about 72 hours; before contacting the cell. The period of from about 30 seconds to about 72 hours may be from about 30, 35, 40, 45, 50, 55 or 60 seconds, 1, 2, 5, 10, 15, 20, 30, 40 or 50 minutes, 1, 2, 3 or 4 hours to about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 35, 36, 40, 44, 48, 52, 56, 60, 64, 68 or 72 hours. The period of from about 30 seconds to about 36 hours may be from about 30, 35, 40, 45, 50, 55 or 60 seconds, 1, 2, 5, 10, 15 minutes to about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 32, 33, 34, 35 or 36 hours.

[0220] In one embodiment of the second, third, fourth, fifth, sixth aspects of the invention, the pre-mix is a defined in the first aspect of the invention, and / or the albumin is as defined in the first aspect of the invention; and / or the retroviridae is as defined in the first aspect of the invention. In one embodiment of the sixth and seventh aspects of the invention, the cell is as defined in the first aspect of the invention; and / or the transduction enhancement is as defined in the first aspect of the invention.

[0221] Also disclosed herein, is a cell free composition comprising an albumin and a retroviridae.

[0222] Otherwise disclosed herein, is a kit of parts comprising an albumin and a retroviridae, wherein the albumin and the retroviridae are cell free.

[0223] In one embodiment of the disclosures above, the composition is a pre-mix as defined in the first aspect of the invention; and / or the albumin is as defined in the first aspect of the invention; and / or the retroviridae is as defined in the first aspect of the invention.

[0224] Otherwise disclosed herein is the composition or the kit disclosed above for use in medicine. The composition or kit disclosed above may be useful in medicine, as the presence of albumin can enhance the transduction of the retroviridae based therapy.

[0225] Otherwise disclosed herein is the composition or the kit disclosed above for use in gene therapy.

[0226] Otherwise disclosed herein is the composition or the kit disclosed above for use in immunotherapy such as oncolytic virotherapy.

[0227] Otherwise disclosed herein is the composition or the kit disclosed above for use as a vaccine.

[0228] Otherwise disclosed herein are the methods, uses, compositions or kits disclosed above wherein the pre-mix does not comprise an anion having multiple negative charges housed on a flexible backbone, such as any of a multivalent carboxylic acid e.g., citrate, fumarate, tartaric acid, a -ketoglutarate, malate, maleic acid, succinate, succinic acid, aconitate, isocitrate, oxaloacetate, adenosine triphosphate or sodium tripolyphosphate or any intermediate anion of the citric acid cycle, or such as succinyl CoA or such as an inorganic anion.

[0229] By the term 'anion having multiple negative charges housed on a flexible backbone' we include the meaning of any flexible backbone that houses two or more negative charges, such as three or more, four or more, or five or more negative charges. Thus, the anion may be multivalent such as divalent, trivalent or tetravalent. In one embodiment, the multiple negative charges housed on a flexible backbone are non- consecutive. In another embodiment, the multiple negative charges housed on a flexible backbone are consecutive. By 'flexible backbone' we include the meaning of a backbone of single covalent bonds that connects the atoms carrying the negatively charged groups, such that there is rotational freedom between the negative charges.

[0230] Examples of suitable anions having multiple negative charges housed on a flexible backbone include multivalent organic anions, such as organic carboxylic acids. For instance, the anion may be a divalent or a trivalent carboxylic acid. In one embodiment, the anion is an intermediate of the citric acid cycle, such as any of citrate, isocitrate, o-ketoglutarate, succinyl CoA, succinate, fumarate, malate, or oxaloacetate. In a further embodiment, the anion is any of tartaric acid, maleic acid, succinic acid, aconitate, adenosine triphosphate or sodium tripolyphosphate. Inorganic anions are also included.

[0231] Otherwise disclosed herein are the methods, uses, compositions or kits disclosed above wherein the pre-mix does not comprise a buffer from the following list: citric acid, tartaric acid, lactic acid or potassium citrate.

[0232] Otherwise disclosed herein are the methods, uses, compositions or kits disclosed above wherein the pre-mix does not comprise a lipopolysaccharide.

[0233] Otherwise disclosed herein are the methods, uses, compositions or kits disclosed above wherein the pre-mix does not comprise a polyethylene glycol (PEG).

[0234] Otherwise disclosed herein are the methods, uses, compositions or kits disclosed above wherein the pre-mix does not comprise 5.0 mM to 20 mM PBS, pH 6.5-7.8, 0.25-25 mM HEPES, 0.01-1 mM MgCI2, 0.01-1 mM CaCI2.

[0235] Otherwise disclosed herein are the methods, uses, compositions or kits disclosed above wherein the pre-mix does not comprise 5.0 mM to 20 mM PBS, pH 6.5-7.8, 0.25-25 mM HEPES, 0.01-1 mM MgCI2, 0.01-1 mM CaCI2, and further does not comprise sucrose, L (+)-glutamic acid, or L (+)-glutamic acid monosodium salt or a mixture of L (+)-glutamic acid / L (+)-glutamic acid monosodium salt. Otherwise disclosed herein are the methods, uses, compositions or kits disclosed above wherein the pre-mix does not comprise a cationic polymer selected from the group consisting of: polyethyleneimine (PEI), polylysine, polyornithine, Polybrene, cyclodextrin, chitosan, histone, collagen, activated and / or non-activated dendrimers.

[0236] Otherwise disclosed herein are the methods, uses, compositions or kits disclosed above wherein albumin is not combined with a pre-formed complex of a lentivirus with PEI (polyethyleneimine).

[0237] Otherwise disclosed herein are the methods, uses, compositions or kits disclosed above wherein albumin is not combined with a pre-formed complex or polyplex of DNA and a cationic polymer selected from the group consisting of: polyethyleneimine (PEI), polylysine, polyornithine, Polybrene, cyclodextrin, chitosan, histone, collagen, activated and / or non-activated dendrimers.

[0238] Otherwise disclosed herein are the methods, uses, compositions or kits disclosed above wherein the pre-mix does not comprise Lenti-X® media (Lenti-X 293T Cell Line growth medium, (Takara Bio Inc)), optionally wherein Lenti-X® media consists essentially of 90% Dulbecco's Modified Eagle's Medium (DMEM), 4.5 g / L of glucose, 4 mM L- glutamine, 3.7 g / L sodium bicarbonate, 10% tetracycline-free fetal bovine serum, and optionally 1 mM sodium pyruvate.

[0239] Otherwise disclosed herein are the methods, uses, compositions or kits disclosed above wherein the pre-mix does not comprise histidine hydrochloride.

[0240] Otherwise disclosed herein are the methods, uses, compositions or kits disclosed above wherein the pre-mix does not comprise one or more of (e.g., all of) a Tris-HCI buffer, B27 serum-free additive and / or CD-Lipid concentrate.

[0241] Otherwise disclosed herein are the methods, uses, compositions or kits disclosed above wherein the pre-mix does not comprise any one or more of the following transduction enhancers: Silibinin, Midostaurin, Amphotericin B, Nystatin, Everolimus, deoxyribonucleosides, BAB-type triblock copolymers such as PEG-PCL-PEG, PEG-PLGA- PEG and PEG-PLA-PEG, Resveratrol, Prostaglandin E2 , Poloxamer Synperonic® F108, Poloxamer 388, Dimethyl sulfoxide (DMSO), Ruxolitinib (Jakavi), Fludarabine (Fludara), Lentiboost® and combinations thereof. Otherwise disclosed herein are the methods, uses, compositions or kits disclosed above wherein the pre-mix does not comprise one or more of HEPES-buffered saline, CaPO4, Tris:EDTA and / or CaCh.

[0242] Otherwise disclosed herein are the methods, uses, compositions or kits disclosed above wherein the method or use in not solely for increasing the stability of the retroviridae.

[0243] It will be appreciated that the method of enhancing transduction of a retroviridae into a cell as described herein may be useful in reducing toxicity of a retroviridae based therapy, for example by minimising or avoiding the need to use known transduction enhancers which may be toxic. Thus, the invention also provides a method of reducing the side effects of a retroviridae based therapy, the method comprising combining the retroviridae with albumin to form a pre-mix; and administering the pre-mix to a subject. Also provided is a method of reducing the side effects of a retroviridae based therapy, the method comprising combining the retroviridae with albumin to form a premix, contacting cells to be transduced with the retroviridae ex vivo, and administering the transduced cells to a subject in need thereof. Preferences for the retroviridae, albumin and pre-mix include those described above in relation to the first aspect of the invention. Preferences for the subject include those described above in relation to the second or third aspects of the invention.

[0244] It will further be appreciated that the method of enhancing transduction of a retroviridae into a cell as described herein may be useful in reducing the dose of a retroviridae therapy (e.g. reducing the number of virus particles administered to the subject and / or reducing the volume of the dose), optionally while maintaining or increasing the efficacy of the therapy. Thus, the method may be used to achieve the desired clinical result using a lower dosing regimen of a retroviridae based therapy. Thus, the invention also provides a method of reducing the dose of a retroviridae based therapy, the method comprising combining the retroviridae with albumin to form a premix, and administering the pre-mix to a subject in need thereof. Also provided is a method of reducing the dose of a retroviridae based therapy, the method comprising combining the retroviridae with albumin to form a pre-mix, contacting cells to be transduced with the retroviridae ex vivo, and administering the transduced cells to a subject in need thereof. Preferences for the retroviridae, albumin and pre-mix include those described above in relation to the first aspect of the invention. Preferences for the subject include those described above in relation to the second or third aspects of the invention. Similarly, it will be appreciated that the method of enhancing transduction of a retroviridae into a cell as described herein may be useful in reducing the overall cost of a retroviridae based therapy, for example achieving the desired clinical result using a lower dosing regimen of a retroviridae based therapy. Thus, the invention also includes a method of reducing the cost of a retroviridae based therapy, the method comprising combining the retroviridae with albumin to form a pre-mix, and administering the pre-mix to a subject in need thereof. Also provided is a method of reducing the cost of a retroviridae based therapy, the method comprising combining the retroviridae with albumin to form a pre-mix, contacting cells to be transduced with the retroviridae ex vivo, and administering the transduced cells to a subject in need thereof. Preferences for the retroviridae, albumin and pre-mix include those described above in relation to the first aspect of the invention. Preferences for the subject include those described above in relation to the second or third aspects of the invention.

[0245] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0246] When we refer to a range, for example a temperature range of "0 to 35°C", we include the meaning of about or approximately 0°C to about or approximately 35°C.

[0247] The listing or discussion in this specification of an apparently prior-published document should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0248] Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the figures.

[0249] EXAMPLE EMBODIMENTS

[0250] 1. A method of enhancing transduction of a retroviridae into a cell, the method comprising:

[0251] (i) combining the retroviridae with albumin to form a pre-mix; and

[0252] (ii) contacting the cell with the pre-mix formed in step (i).

[0253] 2. The method according to Embodiment 1, wherein step (ii) is performed immediately after step (i).

[0254] 3. The method according to Embodiment 1 or 2, wherein step (ii) is performed within a timeframe of from about 30 seconds to about 36 hours after step (i).

[0255] 4. The method according to any one of the preceding embodiments, wherein step (ii) is performed not more than about 30 seconds after step (i), or not more than about 5 minutes after step (i).

[0256] 5. The method according to any one of the preceding embodiments, wherein the pre-mix formed in step (i) is stored at a temperature of from about 0 to about 35°C, or at room temperature, before contacting the cell in step (ii).

[0257] 6. The method according to any one of the preceding embodiments, wherein the pre-mix formed in step (i) is stored at a temperature of from about 15 to about 35°C, or at room temperature, for a period of from about 30 seconds to about 36 hours before contacting the cell in step (ii).

[0258] 7. The method according to any one of Embodiments 1-5, wherein the pre-mix formed in step (i) is stored at a temperature of from about 0 to about 5°C for a period of from about 30 seconds to about 72 hours before contacting the cell in step (ii).

[0259] 8. The method according to any one of Embodiments 1-7 wherein the cell is a dividing cell or a non-dividing cell.

[0260] 9. The method according to any one of Embodiments 1-8 wherein the cell is a eukaryotic cell.

[0261] 10. The method according to Embodiment 9 wherein the cell is an animal cell, fungal cell or a plant cell. 11. The method according to Embodiment 10 wherein the animal cell is a mammalian cell, a fish cell, an insect cell, a reptile cell, an amphibian cell or an avian cell.

[0262] 12. The method according to any one of Embodiments 8-11, wherein the cell is a cell line.

[0263] 13. The method according to Embodiment 12, wherein the cell line is a human embryonic kidney cell line, a T cell (e.g., a human or murine T cell), an immortalised T cell (e.g. a Jurkat cell line), a peripheral blood mononuclear cell (PBMC), a stem cell (e.g. a mesenchymal stem cell (MSC) or a hematopoietic stem cell), a neuron, a cardiomyocyte, a fibroblast, a Chinese hamster ovary (CHO) cell or a HMEC-1 cell.

[0264] 14. The method according to any one of the preceding embodiments, wherein the albumin is sourced from a recombinant source or is sourced from serum.

[0265] 15. The method according to any one of the preceding embodiments, wherein the albumin is a wild-type albumin or a variant thereof, or ovalbumin or a variant thereof.

[0266] 16. The method according to any one of the preceding embodiments, wherein the albumin comprises an amino acid sequence of a mammalian albumin, optionally a human albumin, bovine albumin, dog albumin or mouse albumin.

[0267] 17. The method according to any one of the preceding embodiments, wherein the albumin is:

[0268] (i) recombinant human albumin; or

[0269] (ii) human serum albumin.

[0270] 18. The method according to any one of the preceding embodiments, wherein the albumin is: a) a yeast-derived albumin, optionally wherein the yeast is Pichia such as Pichia pastoris, Saccharomyces such as Saccharomyces cerevisiae, Candida, or Kluyveromyces such as Kluyveromyces lactis or Kluyveromyces marxianus, Hansenula polymorpha, Schizosaccharomyces pombe, Yarrowia lipolytica, Arxula adeninivorans, Candida such as Candida utilis or Zygosaccharomyces such as Zygosaccharomyces bailii; or b) a plant-derived albumin, optionally a rice-derived albumin. 19. The method according to any one of the preceding embodiments, wherein the albumin is a variant of a wild-type albumin, optionally wherein the variant albumin has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the wild-type albumin.

[0271] 20. The method according to any one of the preceding embodiments, wherein the albumin in the pre-mix has a concentration of from about 1 mg / mL to about 400 mg / mL, such as from about 2 to about 400 mg / mL, or from about 2.5 to about 400 mg / mL, from about 3 to about 400 mg / mL, from about 3.5 to about 400 mg / mL, from about 4 to about 400 mg / mL, from about 4.5 to about 400 mg / mL, from about 4.75 mg / mL to about 400 mg / mL, from about 10 mg / mL to about 250 mg / mL, from about 95 mg / mL to about 210 mg / mL, from about 10 mg / mL to about 100 mg / mL, from 2.5 mg / mL to about 100 mg / mL, or from about 7.5 mg / mL to about 50 mg / mL, or from about 10 mg / mL to about 25 mg / mL.

[0272] 21. The method according to any one of the preceding embodiments, wherein following step (ii) the albumin in the composition comprising the pre-mix and the cells has a concentration of from about 0.01 mg / mL to about 4 mg / mL, such as from about 0.01 mg / mL to about 0.025 mg / mL, or from about 0.03 mg / mL to about 0.1 mg / mL, or from about 0.035 mg / mL to about 0.1 mg / mL, or from about 0.04 mg / mL to about 0.1 mg / mL, or from about 0.045 mg / mL to about 0.1 mg / mL or from about 0.005 mg / mL to about 0.075 mg / mL, or from about 0.075 mg / mL to about 0.5 mg / mL, or from about 0.1 mg / mL to about 0.25 mg / mL.

[0273] 22. The method according to any one of the preceding embodiments, wherein the retroviridae is an orthoretrovirinae or spumaretrovirinae, or any virus derived therefrom.

[0274] 23. The method according to Embodiment 22, wherein the orthoretrovirinae is a lentivirus or any virus derived therefrom.

[0275] 24. The method according to Embodiment 22, wherein the orthoretrovirinae is any of an alpharetrovirus, a betaretrovirus, a deltaretrovirus, an epsilonretrovirus, a gammaretrovirus, or any virus derived therefrom.

[0276] 25. The method according to Embodiment 24, wherein the gammaretrovirus is any of a murine leukemia virus, an Abelson murine leukemia virus, a friend virus, a feline leukemia virus, a koala retrovirus (KoRV), a Xenotropic murine leukemia virus-related virus, or any virus derived therefrom.

[0277] 26. The method according to Embodiment 22, wherein the spumaretrovirinae is any of a bovispumavirus, an equispumavirus, a felispumavirus, a prosimiispumarivirus, or a simiispumavirus, or any virus derived therefrom.

[0278] 27. The method according to Embodiment 23, wherein the lentivirus is any of a human immunodeficiency virus (HIV) such as HIV-1 or HIV-2, a simian immunodeficiency virus (SIV), a murine lentivirus, an equine infectious encephalitis virus, an equine infectious anemia virus (EIAV), a caprine arthritis encephalitis virus (CAEV), a bovine immunodeficiency virus (BIV) or a feline immunodeficiency virus (FIV), or any virus derived therefrom.

[0279] 28. The method according to any one of the preceding embodiments, wherein the retroviridae is any of a viral particle, a viral vector such as a retroviridae viral vector, or a vector virus.

[0280] 29. The method according to any one of the preceding embodiments, wherein the retroviridae is a live retroviridae, an attenuated retroviridae, a live-attenuated retroviridae.

[0281] 30. The method according to any one of the preceding embodiments, wherein the retroviridae is in the form of a vaccine.

[0282] 31. The method according to any one of the preceding embodiments, wherein the retroviridae in the pre-mix is present at a multiplicity of infection (MOI) range of 0.1- 100.

[0283] 32. The method according to any one of the preceding embodiments, wherein the pre-mix further comprises a buffer.

[0284] 33. The method according to Embodiment 32, wherein the buffer has a buffering capacity from about pH 6.0 to about pH 8.0.

[0285] 34. The method according to Embodiment 32 or 33, wherein the buffer is any of phosphate-buffered saline (PBS), Hanks buffered salt solution, Earle's balanced salt solution, Tyrode's solution, MOPS buffer, HEPES buffer or any isotonic solutions used for injection, such as Plasma-Lyte®, e.g., Plasma-Lyte® 148, or Ringer's lactate.

[0286] 35. The method according to any one of Embodiments 32-34, wherein the pre-mix comprises 1-250 mM of buffer.

[0287] 36. The method according to any one of the preceding embodiments, wherein the pre-mix comprises from 9.5 to 10.5 mg / mL, preferably about 10 mg / mL, of albumin (w / v), from 20 to 30 mM, preferably from 23 to 26 mM, of sodium, from 0 to 0.03 mM, preferably from 0 to 0.1 mM, of octanoate, wherein the pH is from 6 to 7, preferably about 6.5.

[0288] 37. The method according to any one of the preceding embodiments, wherein the transduction of the retroviridae into a cell is enhanced at least 1.5-fold compared to the transduction of a retroviridae into a cell when the retroviridae is not pre-mixed with albumin.

[0289] 38. The method according to any one of the preceding embodiments, wherein the enhancement of transduction is greater than or equal to the enhancement of transduction provided for by using:

[0290] (i) Lentiboost®; or

[0291] (ii) a transduction enhancer composition comprising Poloxamer 338 or Poloxamer Synperonic® F108, optionally wherein the concentration of Poloxamer 338 or Poloxamer Synperonic® F108 is from about 50 pg / ml to about 5000 pg / ml, such as from about 500 pg / ml to about 1000 pg / ml or from about 50 pg / ml to about 500 pg / ml; or

[0292] (iii) a transduction enhancer composition comprising Polybrene; or

[0293] (iv) a combination of compositions (ii) and (iii).

[0294] 39. The method according to any one of the preceding embodiments, wherein the cell is further contacted with one or more transduction enhancers.

[0295] 40. The method according to any one of the preceding embodiments, wherein the enhanced transduction is manifest by an increase in the expression of a transgene inside the transduced cell, optionally wherein the increase in the expression of the transgene is at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 8.5 times, at least 9 times, at least 9.5 times, or at least 10 times.

[0296] 41. The method according to any one of the preceding embodiments, wherein the enhanced transduction is manifest by an increase in the activity of a protein encoded by a transgene inside the transduced cell.

[0297] 42. A method of improving the efficacy of a retroviridae based therapy, the method comprising combining the retroviridae with albumin to form a pre-mix; and administering the pre-mix to a subject.

[0298] 43. The method according to Embodiment 42, wherein the efficacy is improved due to the enhancement of transduction as a result of combining the retroviridae with albumin to form a pre-mix.

[0299] 44. The method according to Embodiment 42 or 43, wherein the pre-mix is administered to the subject immediately after it was formed.

[0300] 45. The method according to any one of Embodiments 42-44, wherein the pre-mix is administered to the subject within a timeframe of from about 30 seconds to about 36 hours after the pre-mix was formed.

[0301] 46. The method according to any one of Embodiments 42-45, wherein the pre-mix is administered to the subject not more than about 30 seconds after the pre-mix was formed, or not more than about 5 minutes after the pre-mix was formed.

[0302] 47. The method according to any one of Embodiments 42-46, wherein the pre-mix is stored at a temperature of from about 0 to about 35°C, such as from about 0 to about 5°C or from about 15 to about 35°C, or at room temperature before it is administered to the subject.

[0303] 48. The method according to any one of Embodiments 42-47, wherein the pre-mix is stored at a temperature of: i) from about 15 to about 35°C, or at room temperature, for a period of from about 30 seconds to about 36 hours; or ii) from about 0 to about 5°C for a period of from about 30 seconds to about 72 hours before it is administered to the subject. 49. A method of producing a cell therapy ex vivo, wherein the cell therapy comprises cells transduced by a retroviridae, the method comprising:

[0304] (i) providing the cells to be transduced by the retroviridae, and

[0305] (ii) carrying out the method according to any one of Embodiments 1-41 on the cells ex vivo so as to produce the cell therapy.

[0306] 50. A retroviridae based cell therapy for use in in treating a subject, wherein the retroviridae based cell therapy is produced by the method according to Embodiment 49.

[0307] 51. A retroviridae based therapy for use in treating a subject, wherein the retroviridae is combined with albumin to form a pre-mix, which is subsequently administered to the subject.

[0308] 52. The retroviridae based therapy for use according to Embodiment 51, wherein the retroviridae is combined with albumin to form a pre-mix immediately before administration to a subject.

[0309] 53. The retroviridae based therapy for use according to Embodiment 51 or 52, wherein the retroviridae is combined with albumin to form a pre-mix within a timeframe of from about 30 seconds to about 36 hours before administration to a subject.

[0310] 54. The retroviridae based therapy for use according to any one of Embodiments 51-53 wherein the retroviridae is combined with albumin to form a pre-mix not more than about 30 seconds before administration to the subject, or not more than about 5 minutes before administration to the subject.

[0311] 55. The retroviridae based therapy for use according to any one of Embodiments 51-54, wherein the pre-mix is stored at a temperature of from about 0 to about 35°C, such as from about 0 to about 5°C or from about 15 to about 35°C, or room temperature before it is administered to the subject.

[0312] 56. The method according to any one of Embodiments 42-49 or a retroviridae based therapy for use according to any one of Embodiments 51-55, wherein the retroviridae based therapy is an immunotherapy such as oncolytic viral therapy, or wherein the retroviridae based therapy is a gene therapy, or wherein the retroviridae based therapy is a vaccine. 57. Use of albumin to form a pre-mix for enhancing transduction of a retroviridae into a cell, wherein the albumin is combined with the retroviridae to form the pre-mix before contacting the cell.

[0313] 58. Use of a pre-mix for enhancing transduction of a retroviridae into a cell, wherein the pre-mix comprises albumin and the retroviridae and is formed before contacting the cell.

[0314] 59. The use according to Embodiment 57 or 58, wherein the pre-mix is formed immediately before contacting the cell.

[0315] 60. The use according to any one of Embodiments 57-59, wherein the pre-mix is formed within a timeframe of from about 30 seconds to about 36 hours before contacting the cell.

[0316] 61. The use according to any one of Embodiments 57-60, wherein the pre-mix is formed not more than about 30 seconds before contacting the cell, or not more than about 5 minutes before contacting the cell.

[0317] 62. The use according to any one of Embodiments 57-61, wherein the pre-mix is stored at a temperature of:

[0318] (i) from about 0 to about 35°C, or at room temperature for a period of from about 30 seconds to about 72 hours; or

[0319] (ii) from about 15 to about 35°C, or at room temperature, for a period of from about 30 seconds to about 36 hours; or

[0320] (iii) from about 0 to about 5°C for a period of from about 30 seconds to about 72 hours; before contacting the cell.

[0321] 63. The method according to any one of Embodiments 42-49 or 56, the retroviridae based cell therapy according to Embodiment 50 or the retroviridae based therapy for use according to any one of Embodiments 51-56, or the use according to any one of Embodiments 57-62, wherein:

[0322] (i) the pre-mix is a defined in any one of Embodiments 32-36; and / or

[0323] (ii) the albumin is as defined in any one of Embodiments 14-21; and / or

[0324] (iii) the retroviridae is as defined in any one of Embodiments 22-31.

[0325] 64. The use according to any one of Embodiments 57-62, wherein: (i) the cell is as defined in any one of Embodiments 8-13; and / or

[0326] (ii) the transduction enhancement is as defined in any one of Embodiments 37- 40.

[0327] 65. A cell free composition comprising an albumin and a retroviridae.

[0328] 66. A kit of parts comprising an albumin and a retroviridae, wherein the albumin and the retroviridae are cell free.

[0329] 67. The composition according to Embodiment 65, or a kit according to Embodiment 66, wherein:

[0330] (i) the composition is a pre-mix as defined in any one of Embodiments 32-36; and / or

[0331] (ii) the albumin is as defined in any one of Embodiments 14-21; and / or the retroviridae is as defined in any one of Embodiments 22-31.

[0332] 68. The composition according to Embodiment 65 or 67, or a kit according to Embodiment 66 or 67 for use in medicine.

[0333] 69. The composition according to Embodiment 65 or 67, or a kit according to Embodiment 66 or 67 for use in gene therapy.

[0334] 70. The composition according to Embodiment 65 or 67, or a kit according to Embodiment 65 or 67 for use in immunotherapy such as oncolytic virotherapy.

[0335] 71. The composition according to Embodiment 65 or 67, or a kit according to Embodiment 66 or 67 for use as a vaccine.

[0336] 72. The method, use, composition or kit according to any one of the preceding Embodiments wherein the pre-mix does not comprise an anion having multiple negative charges housed on a flexible backbone, such as any of a multivalent carboxylic acid e.g., citrate, fumarate, tartaric acid, a -ketoglutarate, malate, maleic acid, succinate, succinic acid, aconitate, isocitrate, oxaloacetate, adenosine triphosphate or sodium tripolyphosphate or any intermediate anion of the citric acid cycle, or such as succinyl CoA or such as an inorganic anion.

[0337] 73. The method, use, composition or kit according to any one of the preceding Embodiments, wherein the pre-mix does not comprise a buffer from the following list: citric acid, tartaric acid, lactic acid or potassium citrate. 74. The method, use, composition or kit according to any one of the preceding Embodiments, wherein the pre-mix does not comprise a lipopolysaccharide.

[0338] 75. The method, use, composition or kit according to any one of the preceding Embodiments, wherein the pre-mix does not comprise a polyethylene glycol (PEG).

[0339] 76. The method, use, composition or kit according to any one of the preceding Embodiments, wherein the pre-mix does not comprise 5.0 mM to 20 mM PBS, pH 6.5- 7.8, 0.25-25 mM HEPES, 0.01-1 mM MgCI2, 0.01-1 mM CaCI2.

[0340] 77. The method, use, composition or kit according to any one of the preceding Embodiments, wherein the pre-mix does not comprise 5.0 mM to 20 mM PBS, pH 6.5- 7.8, 0.25-25 mM HEPES, 0.01-1 mM MgCI2, 0.01-1 mM CaCI2, and further does not comprise sucrose, L (+)-glutamic acid, or L (+)-glutamic acid monosodium salt or a mixture of L (+)-glutamic acid / L (+)-glutamic acid monosodium salt.

[0341] 78. The method, use, composition or kit according to any one of the preceding Embodiments, wherein the pre-mix does not comprise a cationic polymer selected from the group consisting of: polyethyleneimine (PEI), polylysine, polyornithine, Polybrene, cyclodextrin, chitosan, histone, collagen, activated and / or non-activated dendrimers.

[0342] 79. The method, use, composition or kit according to any one of the preceding Embodiments, wherein albumin is not combined with a pre-formed complex of a lentivirus with PEI (polyethyleneimine).

[0343] 80. The method, use, composition or kit according to any one of the preceding Embodiments, wherein albumin is not combined with a pre-formed complex or polyplex of DNA and a cationic polymer selected from the group consisting of: polyethyleneimine (PEI), polylysine, polyornithine, Polybrene, cyclodextrin, chitosan, histone, collagen, activated and / or non-activated dendrimers.

[0344] 81. The method, use, composition or kit according to any one of the preceding Embodiments, wherein the pre-mix does not comprise Lenti-X® media, optionally wherein Lenti-X® media consists essentially of 90% Dulbecco's Modified Eagle's Medium (DMEM), 4.5 g / L of glucose, 4 mM L-glutamine, 3.7 g / L sodium bicarbonate, 10% tetracycline-free fetal bovine serum, and optionally 1 mM sodium pyruvate. 82. The method, use, composition or kit according to any one of the preceding Embodiments, wherein the pre-mix does not comprise histidine hydrochloride.

[0345] 83. The method, use, composition or kit according to any one of the preceding Embodiments, wherein the pre-mix does not comprise one or more of (e.g., all of) a Tris-HCI buffer, B27 serum-free additive and / or CD-Lipid concentrate.

[0346] 84. The method, use, composition or kit according to any one of the preceding Embodiments, wherein the pre-mix does not comprise any one or more of the following transduction enhancers: Silibinin, Midostaurin, Amphotericin B, Nystatin, Everolimus, deoxyribonucleosides, BAB-type triblock copolymers such as PEG-PCL-PEG, PEG-PLGA- PEG and PEG-PLA-PEG, Resveratrol, Prostaglandin E2 , Poloxamer Synperonic® F108, Poloxamer 388, Dimethyl sulfoxide (DMSO), Ruxolitinib (Jakavi), Fludarabine (Fludara), Lentiboost® and combinations thereof.

[0347] 85. The method, use, composition or kit according to any one of the preceding Embodiments, wherein the pre-mix does not comprise one or more of HEPES-buffered saline, CaPC , Tris: EDTA and / or CaCh.

[0348] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

[0349] Example 1: Albumin enhances the transduction of lentivirus

[0350] Materials and methods

[0351] Cell Lines and Lentivirus

[0352] Human embryonic kidney 293 cells, HEK293, were supplied by the European Collection of Authenticated Cell Cultures (ECACC) and grown in Eagle's Minimum Essential Medium (EMEM, Gibco, UK) supplemented with 10 % fetal bovine serum (FBS, Gibco, USA), 2mM L-glutamine (Gibco, UK), 1% non-essential amino acids (Gibco, UK) and 1 % penicillin / streptomycin (Gibco , USA). Cells were maintained at 37°C, 5% CO2 in a humidified atmosphere. Jurkat cells were supplied by ECACC and grown in RPMI 1640 (Gibco, UK) supplemented with 10% FBS, 2mM L-glutamine (Gibco, UK) and 1 % penicillin / streptomycin (Gibco, USA). Cells were maintained at 37°C, 5% CO2 in a humidified atmosphere. Control Lentiviral particles (product number LV10001, viral concentration was approximately IxlO8lU / ml where "IU" is infectivity units, SEQ ID NO: 6) were supplied by Charles River (USA) and were comprised of the gene for green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter.

[0353] Control Lentiviral particles (product number LV10001) : pLenti-SV40-puro-CMV-GFP- Kan_NGS (SEQ ID NO: 6) :

[0354] GTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTC GTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAG CTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCA GGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATA GTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGG CGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCC TTTTGCTCACATGCTGCTAGAGATTTTCCACACTGACTAAAAGGGTCTGAGGGATCTCTAGTT ACCAGAGTCACACAACAGACGGGCACACACTACTTGAAGCACTCAAGGCAAGCTTTATTGAG GCTTAAGCAGTGGGTTCCCTAGTTAGCCAGAGAGCTCCCAGGCTCAGATCTGGTCTAACCAG AGAGACCCAGTACAGTCCGGATGCAGCTCTCGGGCCATGTGATGAAATGCTAGGCGGCTGT CAAACCTCCACTCTAATACTTCTCTCTCCGGGTCATCCATCCCATGCAGGCTCACAGGGTGTA ACAAGCGGGTGTTCTCTCCTTCATTGGCTTCTTCTACCTTCTCTTGCTCAACTGGTACTAGCTT GTAGCACCATCCAAAGGTCAGTGGATATCTGATCCCTGGCCCTGGTGTGTAGTTCTGCCAAT CAGGGAAGTAGCCTTGTGTGTGGTAGATCCACAGATCAAGGATATCTTGTCTTCGTTGGGAG TGAATTAGCCCTTCCAGTCCCCCCTTTTCTTTTAAAAAGTGGCTAAGATCTACAGCTGCCTTG TAAGTCATTGGTCTTAAAGGTACCTGAGGTGTGACTGGAAAACCCACCTCCTCCTCCTCTTGT GCTTCTAGCCAGGCACAATCAGCATTGGTAGCTGCTGTATTGCTACTTGTGATTGCTCCATGT

[0355] TTTTCTAGGTCTCGATCGAGGTCGACGGTATCGATGCGGGGAGGCGGCCCAAAGGGAGATC

[0356] CGACTCGTCTGAGGGCGAAGGCGAAGACGCGGAAGAGGCCGCAGAGCCGGCAGCAGGCC

[0357] GCGGGAAGGAAGGTCCGCTGGATTGAGGGCCGAAGGGACGTAGCAGAAGGACGTCCCGCG

[0358] CAGAATCCAGGTGGCAACACAGGCGAGCAGCCAAGGAAAGGACGATGATTTCCCCGACAAC

[0359] ACCACGGAATTGTCAGTGCCCAACAGCCGAGCCCCTGTCCAGCAGCGGGCAAGGCAGGCG

[0360] GCGATGAGTTCCGCCGTGGCAATAGGGAGGGGGAAAGCGAAAGTCCCGGAAAGGAGCTGA

[0361] CAGGTGGTGGCAATGCCCCAACCAGTGGGGGTTGCGTCAGCAAACACAGTGCACACCACGC

[0362] CACGTTGCCTGACAACGGGCCACAACTCCTCATAAAGAGACAGCAACCAGGATTTATACAAG

[0363] GAGGAGAAAATGAAAGCCATACGGGAAGCAATAGCATGATACAAAGGCATTAAAGCAGCGT

[0364] ATCCACATAGCGTAAAAGGAGCAACATAGTTAAGAATACCAGTCAATCTTTCACAAATTTTGT

[0365] AATCCAGAGGTTGATTATCGATAAGCTTGATATCGAATTGTACCTAGTGGAACCGGAACCCTT

[0366] AAACATGTATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTCCCTCGACGAATCCTA

[0367] CTTGTACAGACCGCGGCCGGCCGTTTATCTTATCTAGATCCGGTGGATCGGATAAACCTTAT

[0368] TAGTGGTGGTGGTGGTGGTGCTCGACGAATTTATCGTCGTCATCCTTATAATCCTCGACTTAC

[0369] TTGTACAGCTCGTCCATGCCGAGAGTGATCCCGGCGGCGGTCACGAACTCCAGCAGGACCA

[0370] TGTGATCGCGCTTCTCGTTGGGGTCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTT

[0371] GTCGGGCAGCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCGAG

[0372] CTGCACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTTGATGCCGTTCTTCT

[0373] GCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTTGTACTCCAGCTTGTGCCCCAGG

[0374] ATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGATGCGGTTCACCAGGGTGTCGCC

[0375] CTCGAACTTCACCTCGGCGCGGGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCT

[0376] CCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGG

[0377] GTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGTCACGAGGGTGGGCCAGGGCAC

[0378] GGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCC

[0379] TCGCCCTCGCCGGACACGCTGAACTTGTGGCCGTTTACGTCGCCGTCCAGCTCGACCAGGA

[0380] TGGGCACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACCATCTCGAGCGGCCGCGTACG

[0381] CGTCGGTCCGCTAGCTAGTTAAGCTTGAGATCTGGCGCGCCGGCGATCGCGGCGGCAGATC

[0382] TCCTCGGTACCGGATCCAGTCGACGAATTCCCGGCCGCCCTATAGTGAGTCGTATTACAAAA

[0383] TTCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCC

[0384] ATTTGCGTCAACGGGGCGGGGTTATTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCA

[0385] AAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCT

[0386] ATCCACGCCCATTGGTGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACG

[0387] TAGATGTACTGCCAAGTAGGAAAGTCCCGTAAGGTCATGTACTGGGCATAATGCCAGGCGG

[0388] GCCATTTACCGTCATTGACGTCAATAGGGGGCGGACTTGGCATATGATACACTTGATGTACT

[0389] GCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGG

[0390] CGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAG

[0391] CCAGGCGGGCCATTTACCGTAAGTTATGTAACGCGGAACTCCATATATGGGCTATGAACTAA TGACCCCGTAATTGATTACTATTAATAACTAGTCAATAATCAATGTCAACATGGCGGTCATATT GGACATGTCTAGAAATCAGGCACCGGGCTTGCGGGTCATGCACCAGGTGCGCGGTCCTTCG GGCACCTCGACGTCGGCGGTGACGGTGAAGCCGAGCCGCTCGTAGAAGGGGAGGTTGCGG GGCGCGGAGGTCTCCAGGAAGGCGGGCACCCCGGCGCGCTCGGCCGCCTCCACTCCGGGG AGCACGACGGCGCTGCCCAGACCCTTGCCCTGGTGGTCGGGCGAGACGCCGACGGTGGCC AGGAACCACGCGGGCTCCTTGGGCCGGTGCGGCGCCAGGAGGCCTTCCATCTGTTGCTGCG CGGCCAGCCGGGAACCGCTCAACTCGGCCATGCGCGGGCCGATCTCGGCGAACACCGCCC CCGCTTCGACGCTCTCCGGCGTGGTCCAGACCGCCACCGCGGCGCCGTCGTCCGCGACCCA CACCTTGCCGATGTCGAGCCCGACGCGCGTGAGGAAGAGTTCTTGCAGCTCGGTGACCCGC TCGATGTGGCGGTCTGGATCGACGGTGTGGCGCGTGGCGGGGTAGTCGGCGAACGCGGCG GCGAGGGTGCGTACTGCCCGGGGGACGTCGTCGCGGGTGGCGAGGCGCACCGTGGGCTT GTACTCGGTCATGGTCGTACGTGTCTCTTGATCAGATCCGAAAATGGATATACAAGCTCCCG GGAGCTTTTTGCAAAAGCCTAGGCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTCA GAGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGCCATGGGGCGGAG AATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACTATG GTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTT TCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGC CTGGGGACTTTCCACACCCTAACTGACACACATTCTAGAGCGGGCCCGGTACTAACCAAACT GGATCTCTGCTGTCCCTGTAATAAACCCGAAAATTTTGAA I I I I I GTAATTTG I I I I I GTAATT CTTTAGTTTGTATGTCTGTTGCTATTATGTCTACTATTCTTTCCCCTGCACTGTACCCCCCAAT CCCCCCTTTTCTTTTAAAATTGTGGATGAATACTGCCATTTGTCTGCAGAATTGGCGCACGCA

[0392] GTGCCGATCCGTTCACTAATCGAATGGATCTGTCTCTGTCTCTCTCTCCACCTTCTTCTTCTAT TCCTTCGGGCCTGTCGGGTCCCCTCGGGGTTGGGAGGTGGGTCTGAAACGATAATGGTGAA TATCCCTGCCTAACTCTATTCACTATAGAAAGTACAGCAAAAACTATTCTTAAACCTACCAAGC CTCCTACTATCATTATGAATAATTTTATATACCACAGCCAATTTGTTATGTTAAACCAATTCCA CAAACTTGCCCATTTATCTAATTCCAATAATTCTTGTTCATTCTTTTCTTGCTGGTTTTGCGATT CTTCAATTAAGGAGTGTATTAAGCTTGTGTAATTGTTAATTTCTCTGTCCCACTCCATCCAGGT CGTGTGATTCCAAATCTGTTCCAGAGATTTATTACTCCAACTAGCATTCCAAGGCACAGCAGT GGTGCAAATGAGTTTTCCAGAGCAACCCCAAATCCCCAGGAGCTGTTGATCCTTTAGGTATC TTTCCACAGCCAGGATTCTTGCCTGGAGCTGCTTGATGCCCCAGACTGTGAGTTGCAACAGA TGCTGTTGCGCCTCAATAGCCCTCAGCAAATTGTTCTGCTGCTGCACTATACCAGACAATAAT TGTCTGGCCTGTACCGTCAGCGTCATTGACGCTGCGCCCATAGTGCTTCCTGCTGCTCCCAA GAACCCAAGGAACAAAGCTCCTATTCCCACTGCTCTTTTTTCTCTCTGCACCACTCTTCTCTTT GCCTTGGTGGGTGCTACTCCTAATGGTTCAATTTTTACTACTTTATATTTATATAATTCACTTC TCCAATTGTCCCTCATATCTCCTCCTCCAGGTCTGAAGATCAGCGGCCGGCCGCTTGCTGTG CGGTGGTCTTACTTTTGTTTTGCTCTTCCTCTATCTTGTCTAAAGCTTCCTTGGTGTCTTTTAT CTCTATCCTTTGATGCACACAATAGAGGGTTGCTACTGTATTATATAATGATCTAAGTTCTTCT GATCCTGTCTGAAGGGATGGTTGTAGCTGTCCCAGTATTTGTCTACAGCCTTCTGATGTTTCT AACAGGCCAGGATTAACTGCGAATCGTTCTAGCTCCCTGCTTGCCCATACTATATGTTTTAAT

[0393] TTATA I I I I I I CTTTCCCCCTGGCCTTAACCGAA I I I I I I CCCATCGCGATCTAATTCTCCCCC

[0394] GCTTAATACTGACGCTCTCGCACCCATCTCTCTCCTTCTAGCCTCCGCTAGTCAAAATTTTTG

[0395] GCGTACTCACCAGTCGCCGCCCCTCGCCTCTTGCCGTGCGCGCTTCAGCAAGCCGAGTCCT

[0396] GCGTCGAGAGAGCTCCTCTGGTTTCCCTTTCGCTTTCAAGTCCCTGTTCGGGCGCCACTGCT

[0397] AGAGATTTTCCACACTGACTAAAAGGGTCTGAGGGATCTCTAGTTACCAGAGTCACACAACA

[0398] GACGGGCACACACTACTTGAAGCACTCAAGGCAAGCTTTATTGAGGCTTAAGCAGTGGGTTC

[0399] CCTAGTTAGCCAGAGAGCTCCCAGGCTCAGATCTGGTCTAACCAGAGAGACCCAGTACAGG

[0400] CAAAACGCGCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGG

[0401] GGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATT

[0402] GACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTG

[0403] ATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCA

[0404] AGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCAT

[0405] TGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTT

[0406] TACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACA

[0407] TACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTA

[0408] CCGTAAGTTATGTAACGCGGAACTCCATATATGGGCTATGAACTAATGACCCCGTAATTGATT

[0409] ACTATTAATAACTAGTCAATAATCAATGTCAACGCGATATCTGGCCCGTACATCGCGAAGCAG

[0410] CGCAAAACGCCTAACCCTAAGCAGATTCTTCATGCAATTGTCGGTCAAGCCTTGCCTTGTTGT

[0411] AGCTTAAATTTTGCTCGCGCACTACTCAGCGACCTCCAACACACAAGCAGGGAGCAGATACT

[0412] GGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAGGGGATCGGGAGA

[0413] TCTCCCGATCCGTCGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTG

[0414] TTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTT

[0415] CAATAATATTGAAAAAGGAAGAGTATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATT

[0416] AAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAAT

[0417] CAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACAT

[0418] GGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGG

[0419] AATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCAC

[0420] CACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAA

[0421] ATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTC

[0422] CTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTG

[0423] GTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGA

[0424] AATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGAT

[0425] AACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGC

[0426] AGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACA

[0427] GAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTG

[0428] ATGCTCGATGAGTTTTTCTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAA

[0429] AACTTCA I I I I I AATTTAAAAGGATCTAGGTGAAGATCC I I I I I GATAATCTCATGACCAAAAT CCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTT CTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAG CGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGC AGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAA CTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTG GCGATAAGTCGTGTCTTACCGG

[0430] Lentiviral Transduction

[0431] Both HEK293 and Jurkat cells were seeded in a 96 well plate at 1 x 104cells / well and allowed to incubate overnight. In general, for the transductions an MOI of 5 was utilised. Recombinant human serum albumin, Recombumin® Elite (Albumedix, UK) was diluted 1 / 10 in PBS (Gibco, UK) to produce a lOmg / ml solution. Ten microlitres of the diluted albumin or PBS was mixed with the lentivirus to form a pre-mix. The final albumin concentration in the pre-mix was 8.9 mg / ml and 7.1 mg / ml for HEK293 and Jurkat cells respectively. The pre-mix was then added directly the cells. Cells were incubated for 48hrs 37°C, 5% CO2. When required, Lentiboost® was supplied by Sirion Biotech (Germany) and was diluted to 1 : 100 to Img / ml, as a final concentration. Polybrene was supplied by Merck Life Sciences (UK) and used at a final concentration of 8pg / ml. When using Polybrene in transductions with Jurkat cells, after 4 hours incubation the Polybrene was removed by centrifuging the cells at 400 x g for 10 minutes and replacing the Polybrene containing media with complete media, cells were then incubated for 48hrs 37°C, 5% CO2.

[0432] Flow Cytometry Analysis

[0433] Following transduction HEK293 cells were removed from the wells by trypsinization (Gibco, USA), washed and resuspended in BD Pharmingen Stain Buffer (BSA). Jurkat cells were washed and resuspended in stain buffer. For flow cytometry analysis, 10,000 events were analysed for forward and side scatter characteristics and GFP light emission using the FITC filter set (530 / 30) on a BD Celesta™ cell analyzer (BD, USA). The percentage of transduced cells was determined by comparing the GFP expressing transduced cells to non-transduced controls using FlowJo software (Becton Dickinson, USA).

[0434] Statistical Analysis Statistical significance between multiple groups of samples was evaluated by One-way Anova with post-hoc Tukey test. Analysis between 2 groups was performed using unpaired Student's t-test.

[0435] Results and discussion

[0436] Effect of the pre-mix of albumin and Lentivirus on the transduction of HEK293 and Jurkat cells

[0437] The effect of pre-mixing albumin (Recombumin® Elite) with lentivirus immediately before transducing HEK293 and Jurkat cells was examined. The lentivirus and albumin were added to the cells in the following orders: albumin followed by lentivirus, lentivirus followed by the albumin, both lentivirus and albumin added to the cells simultaneously or albumin and lentivirus that had been pre-mixed together before addition to the cells. This was done to establish if the pre-mixing of the lentivirus with albumin was required to achieve the enhanced transduction levels observed (FIG. 1 The final albumin concentration in the pre-mix was 8.9 mg / ml and 7.1 mg / ml for HEK293 and Jurkat cells respectively. Where albumin and lentivirus were added simultaneously rather than as a pre-mix, the resultant albumin concentration was the same as if it had been added in a pre-mix. Figure 1. shows that the albumin and lentivirus need to be mixed together in the form of a pre-mix to achieve any transduction enhancement. When the albumin and lentivirus were added directly to the cells, without prior mixing, the level of transduction found was the same as lentivirus on its own; whereas, when the albumin and lentivirus were combined in a pre-mix, there was approximately a 3-fold increase in transduction for both Jurkat and HEK293 cells.

[0438] Effect of length of time of pre-mix

[0439] The length of time that the pre-mix of albumin and lentivirus could be incubated at room temperature was examined. Stock solutions of albumin and lentivirus or PBS and lentivirus were prepared, incubated at room temperature, and added to the cells at increasing time intervals. After 48 hours of incubation the level of transduction was determined. The percentage of GFP expressing Jurkat cells with the albumin pre-mix remained constant for up to 24 hours (Figure 2), whereas the percentage of GFP expressing cells in the PBS pre-mix declined over time. The number of GFP expressing HEK293 cells did generally decline over time, however, the fold increase in transduction was higher compared to the PBS samples. This data suggests that the pre-mix of albumin and lentivirus could be prepared up to 24 hours in advance and still result in higher levels of transduction.

[0440] Different Concentrations of Recombumin® Elite

[0441] A range of albumin concentrations, l-100mg / ml, were prepared by diluting in PBS. The different albumin solutions were then used in transduction experiments as described above. The Jurkat cells had a significantly higher number of % GFP expressing cells for the highest concentrations of albumin used, 25-100mg / ml (Figure 3), i.e. final albumin concentration in the pre-mix was 17.8-71.4 mg / ml. The number of GFP expressing cells then decreased with albumin concentrations until 2.5mg / ml (i.e. the albumin concentration in the pre-mix was 1.8mg / ml). Concentrations of 2.5mg / ml and Img / ml (i.e. the albumin concentration in the pre-mix was 1.8mg / ml and 0.7 mg / ml, respectively) showed no transduction enhancement as these gave the same number of transduced cells as lentivirus pre-mixed with PBS. For HEK293 cells, the level of transduction showed a similar trend with the higher concentrations of albumin 25-100mg / ml (i.e. the albumin concentration in the pre-mix was 22.3-89.3 mg / ml) giving a higher percentage of GFP expressing cells.

[0442] When testing different concentrations of Recombumin® Elite, by "x mg / ml" we refer to the stock albumin concentration that was used to make the pre-mix. Following mixing with the lentivirus, this resulted in pre-mixes having albumin concentrations according to Table 1.

[0443] Table 1

[0444] A) Jurkat cells B) HEK293 cells

[0445] Effect of Different Source of Human Serum Albumin on Transduction

[0446] Different sources of albumin were tested to see if this had an effect on the level of transduction. Diverse sources of albumin were used, such as recombinant human albumin derived from Saccharomyces cerevisiae (Recombumin® Elite, "Sacch.") human serum albumin ("Serum-derived"), rice derived recombinant albumin ("Rice") and Pichia derived albumin ("Pichia"). All the transductions were performed by creating a pre-mix using an albumin stock solution of 25 mg / ml, resulting in the pre-mix having an albumin concentration of 22.3 mg / mL and 17.8 mg / ml for HEK293 and Jurkat cells respectively. Both cell lines tested show that the source of the albumin did not affect the transduction enhancement (Figure 4). For the Jurkat cells, the fold transduction increase was about 7.5 fold for all samples and for the HEK293, it was about 5 fold for all samples. This indicates that the source of the albumin does not impact the transduction enhancement effect.

[0447] Comparison to alternative albumins

[0448] To determine if the enhanced transduction effect was limited to human sequence albumin, albumins having sequences from different animals were tested. Recombinant dog albumin (SEQ ID NO: 3) and recombinant mouse albumin (SEQ ID NO: 2) were provided as lOOmg / ml solutions and diluted to 25mg / ml with PBS. Chicken egg white albumin or "ovalbumin" (SEQ ID NO: 5) and bovine serum albumin (SEQ ID NO: 4) were supplied as lyophilised powder, stock solutions of 25mg / ml were prepared in PBS. The pre-mix and transduction were performed as described above. The albumin concentration in the pre-mixes was 22.3mg / mL and 17.8mg / mL for HEK293 and Jurkat cells respectively. For the HEK293 cells (Figure 5B), recombinant human albumin (Recombumin® Elite, "Sacch.") and chicken egg white albumin gave similar increases in transduction: 9.3 and 9 fold respectively. Both of these albumins gave higher increases in transduction than the bovine serum albumin, recombinant dog and recombinant mouse albumin which gave 7.8, 7.9 and 7.3 fold increase in transduction respectively. For the Jurkat cells, all of the albumins gave at least 5-fold increase in transduction compared to PBS (Figure 5A).

[0449] Effect of MOI

[0450] Transductions were performed using a range of MOIs, 1-10, to determine if pre-mixing lentivirus with albumin (Recombumin® Elite) still enabled a / the transduction enhancement over a range of lentiviral concentrations. The albumin concentration in the pre-mixes was 8.9mg / mL and 7.1mg / ml for HEK293 and Jurkat cells respectively. For the Jurkat cells, as the MOI increased, the % GFP expressing cells increased for both the lentiviral only samples (PBS) and the albumin-treated samples. However, the fold increase in transduction between the non-albumin treated, and albumin treated samples remained relatively consistent, 4.5, 6.3, 6.2 and 4.2-fold for MOIs of 1, 2, 5 and 10 respectively (Figure 6A). With the HEK293 cells, as the MOI increased, so did the fold increase in transduction between non-albumin treated and albumin treated samples, 1.2, 3.0, 1.8 and 2.4 fold for MOIs of 1, 2, 5 and 10 respectively (Figure 6B).

[0451] Effect of Freezing and Storing the Pre-mix

[0452] To determine if there were any deleterious effects to storing the lentivirus albumin (Recombumin® Elite) pre-mix at -80°C, a pre-mix of lentivirus and albumin was prepared for both HEK293 and Jurkat cells. The albumin concentration in the premixes was 8.9mg / mL and 7.1mg / ml for HEK293 and Jurkat cells respectively. The premix was then divided into 4 aliguots. Three of the aliguots were placed in the -80°C freezer and the remaining aliguot was used in a transduction, this was time 0. One of the remaining aliguots was removed from the freezer on a monthly basis and utilised in a transduction, this was repeated for 3 months. The % GFP expressing cells was determined (Figure 8). For the HEK293 cells at all time points tested, the albumin premix resulted in significantly more GFP expressing cells than the PBS samples. Between time 0 and 1 month there was a drop off in the % GFP expressing cells but this was to be expected as the time 0 samples were not subjected to a freeze thaw cycle, whereas months 1, 2 and 3 all underwent a single freeze thaw cycle. For the months 1, 2 and 3 samples the % GFP expressing cells remained constant for both the PBS and albumin pre-mix samples with the albumin pre-mix samples having significantly more GFP expressing cells for all time points. This data indicates that the benefits of the albumin pre-mix on transduction enhancement can be maintained for up to 6 months in storage at -80°C. The same trend was observed for Jurkat cells (data not shown).

[0453] Comparison to alternative transduction enhancers also lentivirus + albumin combination

[0454] Transductions utilising the albumin (Recombumin® Elite, "Sacch") pre-mix were compared to transductions performed with commercially available transduction enhancers. The commercially available transduction enhancers were utilised according to the manufacturers' instructions. The albumin concentration in the pre-mixes was 22.3 mg / mL and 17.8mg / ml for HEK293 and Jurkat cells respectively. When the commercial transduction enhancer was used in conjunction with the albumin pre-mix, the commercial transduction enhancer was added to the cells followed by the albumin pre-mix. When the albumin pre-mix and the commercial transduction enhancers were combined there was a synergistic effect on the %GFP expressing cells obtained (Figure 7). With the HEK293 cells and Polybrene (Figure 7B), the albumin pre-mix only resulted in 10.6% GFP expressing cells and the Polybrene only 3.8% GFP expressing cells compared to 27.4% for the combination of both Polybrene and albumin pre-mix. This is significantly higher than the combined sum of both of Polybrene and albumin pre-mix alone indicating a synergistic effect. With the HEK293 there was no synergistic effect seen by adding the Lentiboost® in addition to the albumin pre-mix (Figure 7D). The data indicates that the Lentiboost® did not have any effect on increasing the transduction of the lentivirus as the lentivirus-only transduction gave 3.29% GFP expressing cells and the addition of Lentiboost® to the transduction gave 3.06% GFP expressing cells. This result would explain why combining the 2 transduction enhancers had no effect on the level of GFP cells seen, 13.4% for albumin pre-mix only compared to 12.6% for albumin pre-mix and Lentiboost® combined. This result is not significantly different from albumin pre-mix alone indicating that the Lentiboost did not enhance transduction in HEK293 cells.

[0455] With the Jurkat cells, a synergistic effect was seen for both Lentiboost® and Polybrene. With the Lentiboost®, the transduction enhancers alone gave 4.13% and 6.01% GFP expressing cells for albumin pre-mix and Lentiboost® respectively, compared to 23% GFP expressing cells when combined together (Figure 7C). With the Polybrene, the transduction enhancers alone gave 5.55% and 1.17% GFP expressing cells for albumin pre-mix and Lentiboost® respectively, compared to 7.77% GFP expressing cells when combined together (Figure 7A). These results indicate that combining the 2 transduction enhancers has additional advantage when transducing cells.

[0456] Difference between the effect of albumin on transduction of lentivirus and the stability of lentivirus

[0457] To evaluate whether the albumin (Recombumin® Elite) was enhancing the transduction or whether it was improving the stability of lentivirus, two different time course experiments were performed. In one experiment the albumin pre-mix was prepared and incubated at room temperature and samples were taken every 2 hours and added to the cells to perform a transduction; whereas, in the other experiment the lentivirus was stored at room temperature in PBS and mixed with the albumin to form the premix immediately before being added to the cells for the transduction. The albumin concentration in the pre-mixes was 8.9 and 7.1mg / ml for HEK293 and Jurkat cells respectively.

[0458] Lentivirus is unstable at room temperature and the ability of the lentivirus to transduce a cell decreases over time. The lentivirus that is stored in PBS at room temperature will lose its ability to transduce cells over time whereas the lentivirus that is stored in albumin will remain more stable. For both HEK293 and Jurkat cells, the lentivirus stored in PBS showed a decreased transduction efficiency over time compared to the samples stored in albumin (Figure 9). The fold increase in transduction efficiency of pre-mixes comprised of albumin compared to those with PBS increased over time, 2.1 fold to 5.53 fold for Jurkat cells (8 hours) and 2.2 fold to 6.26 fold for HEK293 cells (8 hours) indicating that the albumin stabilised the lentivirus. However, when the lentivirus was stored at room temperature in PBS and then combined with the albumin to form a pre-mix immediately before being added to the cells the effect seen was different (Figure 10). In this experiment, the % GFP expressing cells decreased over time for both albumin and PBS pre-mixes although the albumin pre-mix resulted in a higher % GFP expressing cells than the PBS pre-mix. This indicates that when the lentivirus was mixed immediately with the albumin and used in a transduction the albumin enhanced the transduction rather than increased the stability. For both the Jurkat and HEK293 cells after 4 hours at room temperature, the fold increase in transduction of albumin pre-mix compared to PBS pre-mix remained stable. This indicated that the albumin was not stabilising the virus but enhancing the transduction. When the virus became unstable in PBS at room temperature the albumin was able to rescue the transduction by enhancing transduction rather than stabilising the virus. This is in contrast with the samples stored in the albumin pre-mix which showed an increase in the fold transduction over time (Figure 9). Without wishing to be bound by any theory, this demonstrated that the albumin was not stabilising the virus but enhancing the transduction when combined with the lentivirus immediately before transducing the cells. These two time course experiments illustrate that the effect of albumin on the lentivirus was 2-fold depending on how the albumin was utilised. When the lentivirus was stored in albumin before being added to the cells, albumin both stabilised and enhanced the transduction; however, if the albumin was mixed immediately with the lentivirus and transferred to the cells, it only enhanced the transduction. This work demonstrates the ability of albumin to be used in a novel method to enhance transduction in cells, such as mammalian cells.

[0459] Example 2: Effect of albumin concentration on lentivirus transduction.

[0460] Materials and methods

[0461] Cell Lines and Lentivirus

[0462] Human embryonic kidney 293 cells, HEK293, were supplied by the European Collection of Authenticated Cell Cultures (ECACC) and grown in Eagle's Minimum Essential Medium (EMEM, Gibco, UK) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 2mM L-glutamine (Gibco, UK), 1% non-essential amino acids (Gibco, UK) and 1 % penicillin / streptomycin (Gibco, USA). Cells were maintained at 37°C, 5% CO2 in a humidified atmosphere. Jurkat cells were supplied by ECACC and grown in RPMI 1640 (Gibco, UK) supplemented with 10% FBS, 2mM L-glutamine (Gibco, UK) and 1 % penicillin / streptomycin (Gibco, USA). Cells were maintained at 37°C, 5% CO2 in a humidified atmosphere. Control Lentiviral particles (product number LV10001, viral concentration was approximately IxlO8lU / ml, SEQ ID NO: 6) supplied by Charles River (USA) and were comprised of the gene for green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter.

[0463] Lentiviral Transduction

[0464] Both HEK293 and Jurkat cells were seeded in a 96 well plate at 1 x 104cells / well and allowed to incubate overnight. For the transductions an MOI of 5 was utilised. Recombinant human serum albumin, Recombumin® Elite (Albumedix, UK) was diluted in PBS (Gibco, UK) to produce a range of concentrations from 2mg / ml - 25mg / ml. Ten microlitres of the diluted albumin or PBS was mixed with the lentivirus to form a premix. The albumin concentration in the pre-mixes was 1.4-17.8 mg / ml for Jurkat cells and 1.8 - 22.3 mg / mL for HEK293 cells. The pre-mix was then added directly the cells. Cells were incubated for 48hrs at 37°C, 5% CO2.

[0465] Flow Cytometry Analysis

[0466] Following transduction HEK293 cells were removed from the wells by trypsinization (Gibco, USA), washed and resuspended in BD Pharmingen Stain Buffer (BSA). Jurkat cells were washed and resuspended in stain buffer. For flow cytometry analysis, 10,000 events were analysed for forward and side scatter characteristics and GFP light emission using the FITC filter set (530 / 30) on a BD Celesta™ cell analyzer (BD, USA). The percentage of transduced cells was determined by comparing the GFP expressing transduced cells to non-transduced controls using FlowJo software (Becton Dickinson, USA).

[0467] Statistical Analysis

[0468] Statistical significance between multiple groups of samples was evaluated by One-way anova with post-hoc Tukey test. Analysis between 2 groups was performed using unpaired Student's t-test.

[0469] Results and discussion

[0470] Effect of albumin concentration on the transduction of Lentivirus

[0471] A range of albumin concentrations, 2-25 mg / ml, were prepared by dilution in PBS. The different albumin solutions were then used in transduction experiments as described above. This was to establish at what concentration the albumin started to have a positive effect on the level of transduction observed. For the Jurkat cells there was a significant increase in transduction between the lentivirus only sample and the 4mg / ml albumin sample (Figure 11A). This indicates that at albumin concentrations below 4mg / ml (i.e. the albumin concentration in the pre-mix is 2.9 mg / ml), albumin does not enhance transduction. For the HEK293 cells there was a significant increase in transduction between the lentivirus only sample and the 4.5mg / ml (i.e. the albumin concentration in the pre-mix is 4.0 mg / ml) albumin sample (Figure 11B), demonstrating that at concentrations lower than this the albumin does not enhance transduction. This work is in alignment with the work that was carried out by Palesch et al., (2016). Palesch utilised native HSA as a control in the transduction of a TZM- B1 cell line with HIV and showed that at concentrations of 0 - 100|jg / ml no increase in transduction was observed. In the present Example, we have shown that a stock albumin concentration of at least 4 - 4.5mg / ml (corresponding to an albumin concentration in the pre-mix of 2.9-4.0 mg / ml, and corresponding to -290-400 pg / ml when in the well) is required to achieve transduction enhancement by albumin.

[0472] When testing the effect of albumin concentration on the transduction of Lentivirus, by "x mg / ml" we refer to the stock albumin concentration that was used to make the premix. Following mixing with the lentivirus, this resulted in pre-mix having albumin concentrations according to Table 2.

[0473] Table 2 A) Jurkat cells

[0474] B) HEK293 cells

[0475] Example 3: Albumin enhances the transduction of lentivirus into T cells and Bone marrow derived mesenchymal stem cells (BM-MCS)

[0476] Materials and methods

[0477] Cell Lines and Lentivirus

[0478] Bone marrow derived Mesenchymal stem cells, BM-MSC, were supplied by RoosterBio (USA) and grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco, UK) supplemented with 10% MSC qualified FBS (Gibco, UK). Cells were maintained at 37°C, 5% CO2 in a humidified atmosphere. T cells were extracted from donor blood using the StemCell Technologies EasySep Direct Human T cell Isolation kit (StemCell Technologies, Canada). After extraction the cells were resuspended in Immunocult-XF T cell expansion media (StemCell Technologies, Canada) seeded in a 96 well plate at IxlO5cells / well (in lOOpI media). Cells were then activated with Immunocult Human CD3 / CD28 T cell activator spheres (StemCell Technologies, Canada) and 300 units of IL-2 (StemCell Technologies, Canada). Control Lentiviral particles (product number LV10001, viral concentration was approximately IxlO8lU / ml, SEQ ID NO: 6) were supplied by Charles River (USA) and were comprised of the gene for green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. Lentivira I Transduction

[0479] Bone marrow derived MSCs were seeded into a 96 well plate at 2 x 103cells / cm2and allowed to incubate for 48 hours. In general, for the transductions an MOI of 5 was utilised. Recombinant human serum albumin, Recombumin® Elite (Albumedix, UK) was diluted in PBS (Gibco, UK) to produce a 25mg / ml solution. Ten microlitres of the diluted albumin or PBS was mixed with the lentivirus to form a pre-mix. The albumin concentration in the pre-mix was 17.8mg / ml. The pre-mix was then added directly the cells. Cells were incubated for 72hrs at 37°C, 5% CO2. T cells were activated and seeded as described above. At 48 hours post activation the cells were transduced as described above. Cells were incubated for 48hrs at 37°C, 5% CO2. When required, Lentiboost® was supplied by Sirion Biotech (Germany) and was diluted to 1: 100 to Img / ml, as a final concentration.

[0480] Flow Cytometry Analysis

[0481] Following transduction BM-MSC cells were removed from the wells by trypsinization (Gibco, USA), washed and resuspended in BD Pharmingen Stain Buffer (BSA). T cells were washed and resuspended in stain buffer. For flow cytometry analysis, 5,000 events were analysed for forward and side scatter characteristics and GFP light emission using the FITC filter set (530 / 30) on a BD Celesta™ cell analyzer (BD, USA). The percentage of transduced cells was determined by comparing the GFP expressing transduced cells to non-transduced controls using FlowJo software (Becton Dickinson, USA).

[0482] Statistical Analysis

[0483] Analysis between 2 samples was performed using unpaired Student's t-test.

[0484] Results and discussion

[0485] Effect of the pre-mix of albumin and Lentivirus on the transduction of BM-MSC and T cells

[0486] The effect of pre-mixing albumin (Recombumin® Elite) with lentivirus immediately before transducing BM-MSC and T cells was examined (Figure 12). The lentivirus and albumin were pre-mixed together before addition to the cells. The albumin concentration in the pre-mix was 17.8mg / ml. For the BM-MSC there was approximately 1.5-fold increase in transduction with the pre-mixed sample compared to the lentiviral only control. For the T cells, there was approximately a 6-fold increase in transduction compared to the lentivirus only control. This demonstrates that the method of premixing the albumin has a positive effect on the level of transduction seen for both BM- MSC and T cells.

[0487] Comparison to the alternative transduction enhancers Lentiboost

[0488] Transductions utilising the albumin (Recombumin® Elite, "Sacch") pre-mix were compared to transductions performed with the commercially available transduction enhancer Lentiboost®, which was utilised according to the manufacturer's instructions. The albumin concentration in the pre-mix was 17.8 mg / ml. When the Lentiboost® was used in conjunction with the albumin pre-mix, the Lentiboost® was added to the cells followed by the albumin pre-mix. When the albumin pre-mix and the commercial transduction enhancer were combined there was a positive effect on the % GFP expressing cells obtained (Figure 12). With the BM-MSC cells (Figure 12B) and Lentiboost® the lentivirus only gave 3.72% GFP expressing cells compared to 5.57% and 5.88% GFP expressing cells for albumin and Lentiboost® respectively. When the albumin pre-mix and Lentiboost® were combined together the % GFP expressing cells increased to 9.88%, almost double that of the Lentiboost® transduction enhancer alone. This indicates a strong positive impact on the level of transduction when combining the 2 transduction enhancers in BM-MSC cells.

[0489] With the T cells, a synergistic effect on transduction was observed when combining the albumin pre-mix and the Lentiboost®. With the lentivirus only 1.76% of the cells were transduced. This compares to 10.72% for the T cells transduced with the albumin premix. For Lentiboost®, no increase in transduction was observed when Lentiboost® was used on its own as only 2.03% of cells expressed GFP. However, when the albumin premix and the Lentiboost® were combined for use in transduction, 22.6% of the cells expressed GFP. (Figure 12A). This is a 12.8 fold increase in the level of transduction compared to lentivirus on its own. This demonstrates that with T cells combining the 2 transduction enhancers has a synergistic effect on the level of transduction achieved compared to the individual transduction enhancers.

[0490] Example 4: Albumin enhances the transduction of lentivirus into hematopoietic stem cells

[0491] Materials and methods Cell Line and Lentivirus

[0492] CD34+ cells derived from bone marrow (Stem Cell Technologies, Canada) were grown in StemSpan SFEM medium (Stem Cell Technologies, Canada) supplemented with CC100 supplement (Stem Cell Technologies, Canada). Cells were maintained at 37°C with 5% CO2 in a humidified atmosphere. Control Lentivirus particles were prepared as described in Examples 1-3.

[0493] Lentiviral Transduction

[0494] CD34+ cells were seeded in a 12 well plate at 5000 cells / well (in a volume of 500pl) and allowed to incubate for 24 hours. In general, for the transductions an MOI of 5 was utilised. Recombinant human serum albumin, Recombumin® Elite (Albumedix, UK) was diluted in PBS (Gibco, UK), and to produce a 25 mg / mL solution. Ten microlitres of the diluted albumin or PBS was mixed with lentivirus to form a pre-mix. The albumin concentration in the pre-mix was 18.8 mg / ml. This was then added directly to the cells. At 24 hours post transduction, an additional 500p I of CD34+ complete culture medium was added to the wells and the cells were allowed to incubate for an additional 48 hours.

[0495] Flow Cytometry Analysis

[0496] Post transduction, cells were dissociated from the plate using a cell scraper, washed in BD CellWash (BD, USA) and resuspended in BSA Stain Buffer (BD, USA). For flow cytometry analysis, 10,000 events were analysed for forward and side scatter characteristics and GFP light emission using the FITC filter set (530 / 30) on a BD Celesta™ cell analyzer (BD, USA). The percentage of transduced cells was determined by comparing the GFP expressing transduced cells to non-transduced controls using FlowJo software (Becton Dickinson, USA).

[0497] Statistical Analysis

[0498] Analysis between 2 samples was performed using unpaired Student's t-test.

[0499] Effect of the pre-mix of albumin and Lentivirus on the transduction of CD34+

[0500] CD34+ transductions utilising the albumin (Recombumin® Elite, "Elite") pre-mix were compared to transductions performed in its absence (Figure 13). An increase of approximately 1.5 fold was attained using the Recombumin Elite premix compared to cells transduced with lentivirus alone, with 23.4% GFP positive cells obtained for the albumin pre-mix compared to 15.9% for the lentivirus only. This was shown to be a statistically significant (p<0.01) improvement in lentiviral transduction of CD34+ cells.

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Claims

CLAIMS1. A method of enhancing transduction of a retroviridae into a cell, the method comprising:(i) combining the retroviridae with albumin to form a pre-mix; and(ii) contacting the cell with the pre-mix formed in step (i).

2. The method according to Claim 1, wherein step (ii) is performed :(a) immediately after step (i); or(b) within a timeframe of from about 30 seconds to about 36 hours after step (i); or(c) not more than about 30 seconds after step (i), or not more than about 5 minutes after step (i).

3. The method according to any one of Claim 1 or 2, wherein the pre-mix formed in step (i) is stored at a temperature of:(a) from about 0 to about 35°C, or at room temperature, for a period of from about 30 seconds to about 72 hours before contacting the cell in step (ii); and / or(b) from about 15 to about 35°C, or at room temperature, for a period of from about 30 seconds to about 36 hours before contacting the cell in step (ii); or(c) from about 0 to about 5°C for a period of from about 30 seconds to about 72 hours before contacting the cell in step (ii).

4. The method according to any one of the preceding claims, wherein the cell is:(a) a dividing cell or a non-dividing cell; and / or(b) a eukaryotic cell, optionally wherein the cell is an animal cell, fungal cell or a plant cell, further optionally wherein the animal cell is a mammalian cell, a fish cell, an insect cell, a reptile cell, an amphibian cell or an avian cell; and / or(c) a cell line, optionally wherein the cell line is a human embryonic kidney cell line, a T cell (e.g. a human or murine T cell), an immortalised T cell (e.g. a Jurkat cell line), a peripheral blood mononuclear cell (PBMC), a stem cell (e.g. a mesenchymal stem cell (MSC) or a hematopoietic stem cell), a neuron, a cardiomyocyte, a fibroblast, a Chinese hamster ovary (CHO) cell or a HMEC-1 cell.

5. The method according to any one of the preceding claims, wherein the albumin: (a) is sourced from a recombinant source or is sourced from serum; and / or(b) is a wild-type albumin or a variant thereof, or ovalbumin; and / or(c) comprises an amino acid sequence of a mammalian albumin, optionally a human albumin, bovine albumin, dog albumin or mouse albumin; and / or(d) is:(i) recombinant human albumin; or(ii) human serum albumin; and / or(e) is: i) a yeast-derived albumin, optionally wherein the yeast is Pichia such as Pichia pastoris, Saccharomyces such as Saccharomyces cerevisiae, Candida, or Kluyveromyces such as Kluyveromyces lactis or Kluyveromyces marxianus, Hansenula polymorpha, Schizosaccharomyces pombe, Yarrowia lipolytica, Arxula adeninivorans, Candida such as Candida utilis or Zygosaccharomyces such as Zygosaccharomyces bailii; or ii) a plant-derived albumin, optionally a rice-derived albumin; and / or(f) is a variant of a wild-type albumin or wild-type ovalbumin, optionally wherein the variant albumin or variant ovalbumin has at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the wild-type albumin or wild-type ovalbumin, respectively.

6. The method according to any one of the preceding claims, wherein the albumin in the pre-mix has a concentration of from about 1 mg / mL to about 400 mg / mL, such as from about 2 to about 400 mg / mL, from about 2.5 to about 400 mg / mL, from about 3 to about 400 mg / mL, from about 3.5 to about 400 mg / mL, from about 4 to about 400 mg / mL, from about 4.5 to about 400 mg / mL, from about 4.75 mg / mL to about 400 mg / mL, from about 10 mg / mL to about 250 mg / mL, from about 95 mg / mL to about 210 mg / mL, from about 10 mg / mL to about 100 mg / mL, from about 2.5 mg / mL to about 100 mg / mL, from about 7.5 mg / mL to about 50 mg / mL, or from about 10 mg / mL to about 25 mg / mL.

7. The method according to any one of the preceding claims, wherein following step (ii) the albumin in the composition comprising the pre-mix and the cells has a concentration of from about 0.01 mg / mL to about 4 mg / mL, such as from about 0.01 mg / mL to about 0.025 mg / mL, or from about 0.03 mg / mL to about 0.1 mg / mL, or from about 0.035 mg / mL to about 0.1 mg / mL, or from about 0.04 mg / mL to about 0.1 mg / mL, or from about 0.045 mg / mL to about 0.1 mg / mL or from about 0.005 mg / mL to about 0.075 mg / mL, or from about 0.075 mg / mL to about 0.5 mg / mL, or from about 0.1 mg / mL to about 0.25 mg / mL.

8. The method according to any one of the preceding claims, wherein the retroviridae is an orthoretrovirinae or spumaretrovirinae, or any virus derived therefrom, optionally wherein the orthoretrovirinae is a lentivirus or any virus derived therefrom, further optionally wherein the lentivirus is any of a human immunodeficiency virus (HIV) such as HIV-1 or HIV-2, a simian immunodeficiency virus (SIV), a murine lentivirus, an equine infectious encephalitis virus, an equine infectious anemia virus (EIAV), a caprine arthritis encephalitis virus (CAEV), a bovine immunodeficiency virus (BIV) or a feline immunodeficiency virus (FIV), or any virus derived therefrom.

9. The method according to any one of the preceding claims, wherein the retroviridae in the pre-mix is present at a multiplicity of infection (MOI) range of 0.1- 100.

10. The method according to any one of the preceding claims, wherein the transduction of the retroviridae into a cell is enhanced at least 1.5-fold compared to the transduction of a retroviridae into a cell when the retroviridae is not pre-mixed with albumin.

11. A method of improving the efficacy of a retroviridae based therapy, the method comprising combining the retroviridae with albumin to form a pre-mix; and administering the pre-mix to a subject.

12. A method of producing a cell therapy ex vivo, wherein the cell therapy comprises cells transduced by a retroviridae, the method comprising:(i) providing the cells to be transduced by the retroviridae, and(ii) carrying out the method according to any one of Claims 1-10 on the cells ex vivo so as to produce the cell therapy.

13. A retroviridae based cell therapy for use in in treating a subject, wherein the retroviridae based cell therapy is produced by the method according to Claim 12.

14. A retroviridae based therapy for use in treating a subject, wherein the retroviridae is combined with albumin to form a pre-mix, which is subsequently administered to the subject.

15. Use of albumin to form a pre-mix for enhancing transduction of a retroviridae into a cell, wherein the albumin is combined with the retroviridae to form the pre-mix before contacting the cell.

16. Use of a pre-mix for enhancing transduction of a retroviridae into a cell, wherein the pre-mix comprises albumin and the retroviridae and is formed before contacting the cell.

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