Albuplus-containing retroviral formulations for enhancing transduction efficiency, use thereof for cell transduction

Abstract

This invention relates to methods for enhancing the transduction of retroviral viruses into cells, methods for improving the efficacy of retroviral virus-based therapies, methods for generating cell therapies, and uses thereof. Specifically, this invention relates to a method for enhancing the transduction of retroviral viruses into cells, the method comprising: (i) combining a retroviral virus with albumin to form a premix; and (ii) contacting the cell with the premix formed in step (i).

Description

Technical Field

[0001] This invention relates to methods for enhancing the transduction of viruses, particularly retroviridae viruses, into cells; methods for improving the efficacy of retroviridae virus-based therapies; methods for generating cell therapies in vitro; the use of albumin in the formation of premixes to enhance the transduction of retroviridae viruses into cells; and the use of premixes in enhancing the transduction of retroviridae viruses into cells. Background Technology

[0002] The family Retroviridae, including the subfamilies of Orthoviravins and Retroviravins, is defined by its ability to reverse transcribe an RNA genome into DNA, which integrates into the cellular genome during the viral life cycle within the cell. The family Retroviridae includes retroviruses.

[0003] Retroviruses contain a non-segmented RNA genome, and their hallmark is a replication strategy that involves reverse transcription of viral particle RNA into linear double-stranded DNA (also known as provirus), followed by integration of that DNA into the host genome. Generally, retroviruses enter cells by attaching their surface glycoproteins to specific cell membrane receptors, leading to viral-cell membrane fusion. The provirus is transcribed into mRNA encoding viral proteins, which then package the full-length genomic mRNA into the viral particle to complete the viral life cycle.

[0004] Retroviruses comprise a large and diverse family of enveloped RNAviridae (see below). Retroviruses and lentiviruses belong to the Retroviridae family. Lentivirals are enveloped, single-stranded, positive-sense RNA viruses (Clements & Zink, 1996). Lentivirals 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). The lentiviral genome is delivered to daughter cells of the host as it integrates into the host's DNA. There is a possibility that lentiviruses can mutate / recombine over multiple generations, resulting in infectious, replicating lentiviruses, which could put patients and scientists developing 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 pol) remained together, meaning that only one homologous recombination event was required to restore the vector's replication capacity (Rodrigues et al., 2011). However, modern methods use third-generation lentiviral vectors, which isolate gag, pol, and env on different plasmids (Escors & Breckpot, 2010), meaning that at least three homologous recombination events are required to restore replication capacity, which is unlikely to occur.

[0005] The ability of viruses to infect and genetically modify human cells can be therapeutically used in gene therapy, such as for the treatment of β-thalassemia, hemophilia, and adrenoleukodystrophy (Vargas et al., 2016). Retroviral vector technology is the most commonly used method for gene transfer in gene therapy. Two general approaches exist: in vivo gene therapy, where genes are delivered directly to target cells; and ex vivo therapy, where target cells are genetically modified in vitro and then transplanted into a subject.

[0006] In cell-modified gene therapy, lentiviruses are commonly used as vectors to introduce transgenes into cells, a process known as transduction. Transduction is the virus-mediated transfer of nucleic acids into cells. Viruses are able to infect cells and transport nucleic acids into the cell nucleus without further action. Transduction can be transient, where nucleic acids remain free, or it can be stable, where nucleic acids integrate into the cell's genome. For example, lentiviruses have the ability to permanently integrate into the cell's genome. After the virus enters the cell, viral RNA is transcribed by reverse transcriptase to produce double-stranded DNA that enters the cell nucleus. Transgenes are integrated into the host genome via lentiviral integrase. Once a cell has been transduced by lentiviral particles, the cell expresses transcripts and / or proteins of the gene of interest.

[0007] Despite several clinical trials of retroviral-based therapies, success has been limited. A key challenge in retroviral-based therapies is the need for viral particles with high multiples of infection (MOI) to provide sufficient transgene integration and expression. Various approaches are currently used to help increase viral transduction, such as increasing viral titers to increase MOI, prolonging the duration of viral infection, and / or repeating multiple rounds of viral infection. This can lead to increased side effects and the risk of off-target effects.

[0008] Other approaches to ensure adequate transgene expression include overexpressing viral receptors (e.g., PIT1 and PIT2) on the cells to be transduced and / or optimizing the viral vector cassette using strong gene promoters and / or gene enhancers, codon optimization of the transgene cDNA, efficient polyadenylation sequences, and the use of a self-complementary vector genome. However, such methods are generally not suitable for all viral vectors and / or transgenes.

[0009] Transduction enhancers are commonly used to assist transduction. These enhancers work by assisting viral transduction, resulting in a higher proportion of cells being transduced, or even reducing MOI while maintaining the same transduction level. Polybrene, a transduction enhancer for retrovirid viruses, is a polycation believed to neutralize the electrostatic repulsion between the cell lipid bilayer and the viral lipid bilayer, thus promoting viral binding to the cell surface (Andreadis & Palsson, 1997). Polybrene is a popular enhancer choice due to its low cost and ability to improve transduction efficiency (Davis et al., 2002); however, it can be cytotoxic if used at high concentrations. Another transduction enhancer is called Lentiboost. ® This transduction enhancer contains poloxamer 338, which can be defined as a “large nonionic amphiphilic molecule” (Lee et al., 1992), and is thought to increase transduction by interacting with the cell membrane to allow genetic material to pass through (Lee et al., 1992); however, this commercial product is expensive.

[0010] Therefore, there is an unmet need for safer and / or less expensive lentiviral transduction enhancers.

[0011] Albumin is a key protein found in blood and accounts for 60% of the total protein content in serum (Francis, 2010). Albumin is a heart-shaped polypeptide molecule with a molecular weight of 66.5 kDa (Sand et al., 2014). It is a non-glycosylated helical molecule composed of 585 amino acids (Horváthy et al., 2017). Albumin has many biological functions; a notable function is that it is an antioxidant, meaning it is available in reducing oxidative stress within cells. This is due to the reduction of the free thiol group found on cysteine ​​residue -34, which means that albumin can scavenge free radicals (Francis, 2010). Albumin is also known to bind directly to the cell surface in a non-specific manner to “protect the cell,” making it biomedically usable (Yamazoe and Tanabe, 2008). Thus, albumin can be used in medical devices to prevent biofilm formation. Another key function of albumin is its ability to bind to fatty acids, lipids, and metal ions to help influence cellular processes and its use as a key component in cell culture growth media. Fetal bovine serum (FBS) is a standard additive in cell culture media; however, human serum albumin (HSA) has become a popular choice due to the demand for animal-free media in cell and gene therapy. Furthermore, researchers are turning to recombinant albumin sources due to supply chain issues and regional variations in HSA.

[0012] Wang et al. (2017) found that pre-incubating adeno-associated virus (AAV) with HSA increased the transduction efficiency of the virus. The enhanced transduction efficiency observed by Wang et al. (2017) was thought to work through a direct interaction between albumin and the AAV capsid. However, lentiviruses differ structurally from AAVs due to the presence of a lipid envelope surrounding the lentiviral capsid. Therefore, it was not expected that albumin could enhance lentiviral transduction in the same way as with AAVs. Surprisingly, however, the inventors have demonstrated that albumin can enhance lentiviral transduction in at least human embryonic kidney (HEK) 293 cells and Jurkat cells. Specifically, premixing albumin and lentivirus before adding them to the cells was found to enhance lentiviral transduction, making it possible to reduce the MOI while maintaining the same transduction level. The inventors also found that combining known transduction enhancers with albumin showed at least a 2-fold increase in transduction efficiency (i.e., at least double) compared to using transduction enhancers alone. Summary of the Invention

[0013] The present invention relates to a method for enhancing the transduction of retroviral viruses into cells, the method comprising: (i) combining the retroviral virus with albumin to form a premix; and (ii) contacting the cell with the premix formed in step (i).

[0014] The present invention also relates to a method for improving the efficacy of a therapy based on a retroviridae virus, the method comprising combining the retroviridae virus with albumin to form a premix; and administering the premix to a subject.

[0015] The present invention also relates to a method for generating cell therapy in vitro, wherein the cell therapy comprises cells transduced by a retroviridae virus, the method comprising: (i) providing the cells to be transduced by the retroviridae virus, and (ii) subjecting the cells in vitro to the method of claim 1 to generate the cell therapy.

[0016] The present invention also relates to a retroviral virus-based cell therapy for treating subjects.

[0017] The present invention also relates to a method of treating a subject, the method comprising administering a retroviral virus-based cell therapy to the subject in need, wherein the method comprises (a) forming a premix; (b) adding the premix to the cells; and (c) administering the transduced cells to the subject.

[0018] The present invention further relates to a retroviridae virus-based therapy for treating a subject, wherein the retroviridae virus is combined with albumin to form a premix, and the premix is ​​subsequently administered to the subject.

[0019] The present invention also relates to a method of treating a subject, the method comprising administering a retroviral virus-based therapy to a subject in need, wherein the method comprises (a) forming a premix; and (b) administering the premix to the subject.

[0020] The present invention also relates to the use of albumin in the formation of a premix to enhance the transduction of retroviridae viruses into cells, wherein the albumin is combined with the retroviridae virus to form the premix prior to contact with the cell.

[0021] The present invention further relates to the use of a premix for enhancing the transduction of retroviridae viruses into cells, wherein the premix comprises albumin and the retroviridae virus, and the premix is ​​formed prior to contact with the cells. Attached Figure Description

[0022] Figure 1 - Effect of albumin and lentivirus premixes on transduction efficiency of (A) Jurkat cells and (B) HEK293 cells. For the premix, both albumin and lentivirus were added to Eppendorf tubes and vortexed briefly, e.g., 2–3 seconds. For "albumin then lentivirus," albumin was pipetted into wells, followed by lentivirus. For "lentivirus then albumin," lentivirus was pipetted into wells, followed by albumin. For "together but separately," albumin and lentivirus were pipetted into wells simultaneously. Transduction efficiency was measured as GFP% by the FITC channel on a FACS Celesta flow cytometer.

[0023] Figure 2 - The effect of the length of time the premix was incubated on the transduction efficiency of (A) Jurkat cells and (B) HEK293 cells. Incubation was performed at room temperature and ranged from immediately (within seconds) before the premix was added to the cells to 24 hours.

[0024] Figure 3 - Investigating different concentrations of albumin (Recombinant albumin) in a premix of albumin and lentivirus. ® Elite (to determine the optimal concentration / concentration range that provides maximum transduction efficiency in (A) Jurkat cells and (B) HEK293 cells). Albumin concentration refers to the stock albumin concentration. For the final albumin concentration in the premix, refer to Example 1.

[0025] Figure 4- The effect of albumin from different sources on transduction. Different recombinant albumins and human serum albumin at concentrations of 25 mg / mL were used in the premix (the final albumin concentrations in the premix were 17.8 mg / mL and 22.3 mg / mL for Jurkat cells and HEK293 cells, respectively, when diluted with lentivirus) and then used to transduce (A) Jurkat cells and (B) HEK293 cells.

[0026] Figure 5 - Comparison of alternative sources of albumin (animal species sequences) for transduction. Different albumins at a concentration of 25 mg / mL were used in the premix for transduction of (A) Jurkat cells and (B) HEK293 cells.

[0027] Figure 6 - The effect of MOI on transduction enhancement. A series of MOIs were detected to determine the effect of lentivirus on albumin (Recombumin). ® Does the effect of premixing (Elite) still enhance transduction in (A) Jurkat cells and (B) HEK293 cells at different amounts of lentivirus?

[0028] Figure 7 - Combination of commercially available transduction enhancers and albumin (Recombumin) ® The effect of the Elite premix. A and B show data for Jurkat and HEK293 cells obtained with the transduction enhancer polybrene, respectively. C and D show data for cells obtained with the transduction enhancer Lentiboost, respectively. ® Data obtained from Jurkat and HEK293 cells.

[0029] Figure 8 - Freezing and storing albumin (Recombumin) ® Effects of the Elite premix. Premix samples were prepared at time 0, aliquoted and used immediately for transduction assays, or stored at -80°C and used monthly for transduction assays over a period of 3 months in HEK293 cells.

[0030] Figure 9 - Lentiviral virus was stored at room temperature with albumin (Recombumin) before transducing (A) Jurkat cells and (B) HEK293 cells. ® The effect of Recombumin Elite premix on transduction efficiency. The figures represent the fold increase in transduction with the Recombumin Elite premix compared to the PBS premix.

[0031] Figure 10- Store lentivirus in PBS at room temperature, then mix with albumin (Recombinant human lysate). ® The effect of combining Recombumin Elite premixes to form a premix and immediately adding it to (A) Jurkat cells and (B) HEK293 cells on transduction efficiency. Figures represent the fold increase in transduction with the Recombumin Elite premix compared to the PBS premix. The fold increase in transduction in Jurkat cells over 4–10 hours and in HEK293 cells over 4–8 hours was not statistically significant.

[0032] Figure 11 - The effect of different concentrations of albumin on the enhanced transduction of lentivirus into Jurkat cells (A) and HEK293 cells (B). Albumin concentration refers to the concentration of the stock albumin. For the final albumin concentration in the premix, refer to Example 2.

[0033] Figure 12 - The use of albumin (“Sacch.”) in enhancing lentiviral transduction of T cells (A) and BM-MSCs (B). The figure also shows the commercially available transduction enhancer Lentiboost. ® Effects of albumin alone and in combination.

[0034] Figure 13 - The use of albumin (“Elite”) in enhancing lentiviral transduction of CD34+ cells. Detailed Implementation

[0035] In a first aspect, the present invention provides a method for enhancing the transduction of a retroviridae virus into a cell, the method comprising: (i) combining the retroviridae virus with albumin to form a premix; and (ii) contacting the cell with the premix formed in step (i).

[0036] The terms "enhanced transduction," "enhanced transduction," or "strengthened transduction" include the following meanings: an increased ability of the virus to transduce cells, for example, relative to the ability of the virus to transduce cells when it has not yet been premixed with albumin. We also include the following meaning: increased transduction efficiency.

[0037] Methods for assessing the ability of viruses to transduce cells are well known in the art, and any suitable method can be used. Examples of methods are described below and in the embodiments.

[0038] Conveniently, viruses contain detectable nucleic acids in their genomes, which can be detected intracellularly once the virus has transduced a cell. Detectable nucleic acids can be directly detectable. For example, nucleic acids can be detected after hybridization with a probe containing a detectable motif (e.g., a fluorophore or radiolabel). Nucleic acids can also be detected indirectly, for example, after the expression of a protein encoded by the nucleic acid, which can be detected, for example, by assessing its expression or activity. It should be understood that the viral genome can be modified to contain detectable nucleic acids, which can be used as so-called “reporter genes” to measure transduction levels. In this work, the reporter gene green fluorescent protein (GFP) was used as a transgene, and GFP expression in cells was monitored to determine the level of transduction enhancement. Other suitable reporter genes are known in the art and include yellow fluorescent protein, lacZ, alkaline phosphatase, and firefly luciferase.

[0039] In one embodiment, the transduction of retroviral viruses into cells is enhanced by at least 1.01-fold compared to the transduction of retroviral viruses into cells when they are not premixed with albumin, for example, by at least 1.02-fold, 1.03-fold, 1.04-fold, 1.05-fold, 1.1-fold, 1.2-fold, and 1... 3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3.0x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4.0x, 4.1x, 4x. 2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, 4.9x, 5.0x, 5.1x, 5.2x, 5.3x, 5.4x, 5.5x, 5.6x, 5.7x, 5.8x, 5.9x, 6.0x, 6.1x, 6.2x, 6.3x, 6.4x, 6.5x, 6.6x, 6.7x, 6.8x, 6.9x, 7.0x, 7.1x 7.2x, 7.3x, 7.4x, 7.5x, 7.6x, 7.7x, 7.8x, 7.9x, 8.0x, 8.1x, 8.2x, 8.3x, 8.4x, 8.5x, 8.6x, 8.7x, 8.8x, 8.9x, 9.0x, 9.1x, 9.2x, 9.3x, 9.4x, 9.5x, 9.6x, 9.7x, 9.8x, 9.9x, or 10x. The term "fold change" includes changes in the percentage of successfully transduced cell populations. "1x" means no change in the percentage of successfully transduced cell populations, and (for example) a 1.05x enhancement means a 5% increase in the percentage of successfully transduced cell populations.

[0040] In one embodiment, enhanced transduction manifests as an increase in transduced transgene expression, optionally wherein the increase in transgene expression is at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4.0-fold, 4.1-fold, 4.2-fold, 4.3-fold, 4.4-fold, 4.5-fold, 4.6-fold, or 4.7-fold compared to transgene expression when the retrovirid virus is not premixed with albumin. 4.8 times, 4.9 times, 5.0 times, 5.1 times, 5.2 times, 5.3 times, 5.4 times, 5.5 times, 5.6 times, 5.7 times, 5.8 times, 5.9 times, 6.0 times, 6.1 times, 6.2 times, 6.3 times, 6.4 times, 6.5 times, 6.6 times, 6.7 times, 6.8 times, 6.9 times, 7.0 times, 7.1 times, 7.2 times, 7.3 times, 7. 4 times, 7.5 times, 7.6 times, 7.7 times, 7.8 times, 7.9 times, 8.0 times, 8.1 times, 8.2 times, 8.3 times, 8.4 times, 8.5 times, 8.6 times, 8.7 times, 8.8 times, 8.9 times, 9.0 times, 9.1 times, 9.2 times, 9.3 times, 9.4 times, 9.5 times, 9.6 times, 9.7 times, 9.8 times, 9.9 times, or 10 times. As an example, in this text, expressing an increase of "1." times" means an increase of 50%.

[0041] The term "genetically modified" includes the inclusion of foreign nucleic acids in the genome of viruses belonging to the Retroviridae family. For example, a genetically modified organism (GMO) can be a reporter gene, a biomarker, and / or a therapeutic gene. However, it should be understood that a genetically modified organism does not necessarily need to include a reporter gene or a biomarker.

[0042] Transgenic genes can contain any gene of interest, such as nucleic acid sequences encoding proteins that are intended to be integrated into the genome of retroviridae viruses. Expression of the transgene can be transient or stable / integrated. Conveniently, the gene of interest can be operatively linked to one or more other sequences that can be used to obtain 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 marmot 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-1α1, MND, MCU3, SFFV, and CBh) or tissue-specific promoters (such as CD11b, ALB, TBG, MHC, MLC2v, and eTnT promoters). In the case of therapeutic transgenes, tissue-specific promoters can be used to ensure that the gene expression level is consistent with the expression of the “damaged” or missing gene that the transgene is intended to replace / enhance.

[0043] In one implementation, the transgene encodes a protein reporter gene. The protein reporter gene 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 gene (such as LacZ (β-galactosidase)), an antibiotic resistance marker (such as a puromycin resistance marker), or a combination thereof.

[0044] It should be understood that transgenics can encode more than one gene of interest. For example, transgenics can encode therapeutic proteins as well as reporter proteins and / or biomarker proteins. The presence of a reporter gene or biomarker can be used to monitor or detect the expression of a therapeutic protein in a subject. However, it is also recognized that the expression of a therapeutic protein can be detected without the use of a reporter molecule or biomarker molecule, therefore, transgenics need not also contain a reporter gene or biomarker.

[0045] In one implementation, enhanced transduction manifests as increased expression of one or more RNA species within the transduced cell. Examples of RNA species may include mRNA, short interfering RNA, small hairpin RNA, short guide RNA, long noncoding RNA, or microRNA.

[0046] The term “expression” of a transgenic or RNA species includes the meaning of one or more of the following events: (1) reverse transcription of viral RNA into DNA, (2) integration of viral DNA into the host genome, (3) generation of an RNA template from a DNA sequence (e.g., by transcription); (4) processing of RNA transcripts (e.g., by splicing, editing, 5' cap formation and / or 3' end processing); (5) translation of RNA into a polypeptide or protein; (6) folding of a polypeptide or protein; and (7) post-translational modification of a polypeptide or protein.

[0047] Those skilled in the art will be able to measure the expression of transgenes. For example, RNA templates or transcripts can be detected, for example, using polymerase chain reaction, and quantified, for example, using real-time (RT) quantitative PCR (qPCR), quantitative reverse transcription PCR, digital PCR, or digital droplet PCR; peptides or proteins can be detected, for example, using immunohistochemistry, and quantified, for example, using flow cytometry, Western blotting, or enzyme-linked immunosorbent assay (ELISA).

[0048] In another implementation, enhanced transduction manifests as increased activity of the transgene-encoded protein within the transduced cells.

[0049] It should be understood that different proteins have different functions, such as: enzymes (e.g., amylase, lipase, pepsin), transporters (e.g., hemoglobin), structural supporters (e.g., actin, tubulin, keratin), chemical signal transduction (e.g., insulin), and immune responses (e.g., antibodies). The term "protein activity" includes the meaning of a protein performing its known function. The term "increased activity" includes the meaning of a protein performing its known function better or at a higher level. For example, an enzyme may process more substrate into a product, an increase in product can be detected, and so on.

[0050] Those skilled in the art will be able to use any suitable method to measure the transduction of retroviridae viruses into cells. For example, retroviridae viruses may contain a reporter gene or a marker (such as a fluorescent marker or an antibiotic resistance marker). The number of fluorescent cells or antibiotic-resistant cells in a population can then be quantified. For example, flow cytometry using fluorescent protein markers (e.g., fluorescence-activated cell sorting (FACS)) or antibiotic selection can be used to determine the percentage of infected cells. In a particular example, a retroviridae virus particle may contain a gene encoding a reporter gene or marker (e.g., a fluorescent protein) under promoter control, such that when the retroviridae virus is transduced into cells, the gene is expressed and a reporter gene or marker (e.g., a fluorescent protein) is produced. A plate reader can be used to measure the bioluminescence of the luminescent marker. Live-cell imaging (such as Incucyte) ®The live cell analysis system (Sartorius) can be used to measure fluorescent proteins.

[0051] The transduction level of a virus can be expressed as transduction efficiency, which is the proportion of cells that are successfully transduced once the virus comes into contact with a cell population. Therefore, the so-called enhanced viral transduction includes the following meanings: increased viral transduction efficiency, for example, involving the viral transduction efficiency when the virus has not yet been premixed with albumin.

[0052] The term "premix" encompasses a composition containing a retroviral virus and albumin. The retroviral virus and albumin components of the premix are mixed together before contact with the cells to be transduced; therefore, it should be understood that the composition does not contain the cells to be transduced. Thus, the premix is ​​typically cell-free. "Cell-free" means that the composition is substantially free of cells, particularly the cells to be transduced. Mixing may or may not include agitation, such as shaking, oscillation, flicking, oscillation in generally circular motions (e.g., vortexing), or repeated pipetting (i.e., by repeatedly distributing and aspirating the solution, typically up and down). Preferably, the premix is ​​formed by oscillation, such as vortexing for 1-4 seconds, for example 2-3 seconds.

[0053] In a preferred embodiment, step (ii) is performed immediately after step (i). "Immediately after" includes the following meaning: contacting the cells with the premix within 3, 5, 10, 15, 20, 25, or 30 seconds of combining the retroviridae virus with albumin to form the premix. For example, step (ii) can be performed within 1 to 30 seconds of combining the retroviridae virus with albumin. Preferably, step (ii) is performed immediately after step (i) or within a time range of approximately 0 hours (immediately) to approximately 2 hours, such as approximately 1 second, 5 seconds, 10 seconds, 20 seconds, or 30 seconds to approximately 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 60 minutes.

[0054] In another implementation, step (ii) is performed over a time period of approximately 30 seconds to approximately 36 hours after step (i), such as over a time period of approximately 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds or 60 seconds to approximately 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 28 hours, 32 hours, 33 hours, 34 hours, 35 hours or 36 hours, such as over a time period of approximately 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes or 60 minutes.

[0055] In one implementation, step (ii) is performed no more than about 30 seconds after step (i), or no more than about 5 minutes after step (i), such as no more than about 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds or 60 seconds, no more than about 1 minute, 2 minutes, 3 minutes, 4 minutes or 5 minutes.

[0056] In one embodiment, the premix formed in step (i) is stored at a temperature of about 0°C to about 35°C (°C) or at room temperature, such as about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C or 20°C to about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C, and then contacted with cells in step (ii).

[0057] The term "storage" includes the following meanings: incubating the premix at a non-freezing temperature (such as 0°C to 35°C) for any period of time, provided that the retroviridae virus is still able to transduce within the cell. The term "room temperature" includes a temperature of approximately 21°C.

[0058] In one embodiment, the premix formed in step (i) is stored at a temperature of about 15°C to about 35°C or at room temperature, such as about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C or 25°C to about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C, for about 30 seconds. The time period is approximately 36 hours (e.g., approximately 30 seconds to approximately 24 hours), such as approximately 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds or 60 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes to approximately 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 28 hours, 32 hours, 33 hours, 34 hours, 35 hours or 36 hours, and then the cells are contacted in step (ii).

[0059] In another embodiment, the premix formed in step (i) can be stored at a temperature of at least 35°C to about 47°C, such as 35°C, 35.5°C, 36°C, 36.5°C, 37°C, 37.5°C, 38°C, 38.5°C, 39°C, 39.5°C or 40°C to 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C or 47°C, for a period of time from 30 seconds to 8 hours, such as 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds or 60 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes to about 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and then contacted with cells in step (ii).

[0060] In another embodiment, the premix formed in step (i) is stored at a temperature of about 0°C to about 5°C, such as about 0°C, 0.5°C, 1.0°C, 1.5°C, 2.0°C, 2.5°C, or 3.0°C to about 2.0°C, 2.5°C, 3.0°C, 3.5°C, 4.0°C, 4.5°C, or 5°C, for a period of about 30 seconds to about 72 hours, such as about 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, or 60 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 4... The time interval is 5 minutes or 60 minutes, 1 hour, 2 hours or 3 hours to approximately 30 minutes, 45 minutes, 60 minutes, or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, or 72 hours, and then the cells are contacted in step (ii).

[0061] In a preferred embodiment, steps (i) and (ii) are performed at room temperature, and step (ii) is performed immediately after step (i).

[0062] As shown in Example 1, the inventors have demonstrated that when the premix is ​​formed in step (i) and immediately frozen, and then thawed before proceeding to step (ii), the enhancement of transduction remains significant. Therefore, in one embodiment, the premix is ​​frozen (e.g., at about -15°C to about -90°C, for example, at about -15°C to about -25°C, for example, at about -70°C to about -90°C, for example, at about -20°C or about -80°C) and thawed (e.g., at about 2°C to about 6°C, for example, at about 4°C or at about room temperature), and then contacted with cells in step (ii). The premix can be frozen (e.g., at about -70°C to about -90°C, such as 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), then thawed and contacted with cells in step (ii).

[0063] In one embodiment, step (i) further includes a freeze-thaw cycle. A "freeze-thaw cycle" means that the formulation is frozen at about -15°C to about -90°C, for example, at about -15°C to about -25°C, or for example, at about -70°C to about -90°C, preferably at about -20°C or about -80°C, for a set period of time (e.g., at least 19 hours), and thawed at room temperature for a set period of time (e.g., at least 5 hours), and then frozen again and the cycle is repeated.

[0064] The cell can be any cell known to be transduced by or capable of being transduced by retroviridae viruses (e.g., retroviruses or lentiviruses). 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 a avian cell.

[0065] Different subclasses of the Retroviridae family may have different abilities to transduce different cell types. For example, lentiviruses can transduce both dividing and non-dividing cells, while another subclass of the Retroviridae family, gamma retroviruses, can transduce only dividing cells. This is because they can only enter the nucleus during mitosis, when the nuclear membrane is disrupted.

[0066] Therefore, it should be understood that cells can be dividing or non-dividing cells. The term "dividing cell" includes cells that are undergoing cell division (such as mitosis or meiosis) or (in their current state) are capable of undergoing cell division. Such dividing cells can be totipotent or pluripotent and include stem cells, such as embryonic stem cells, adult stem cells, or stem cells in plant meristems. Some cells (e.g., CD34+ stem cells) are "slow" dividing cells. The term "non-dividing cell" includes cells that are not undergoing cell division or (in their current state) are incapable of undergoing cell division, such as terminally differentiated cells. Such non-dividing cells can include muscle cells, such as cardiomyocytes; primary neurons; glial cells; lymphocytes; macrophages; dendritic cells; epithelial cells; and adipocytes.

[0067] Under certain conditions, dividing cells can become non-dividing cells by entering the inactive phase of mitosis (G0 phase, also known as the quiescent phase). Under certain conditions, non-dividing cells can become dividing cells.

[0068] In one embodiment, the cell is a cell line. The term "cell line" includes the meaning of a defined population of cells that can be maintained for an extended period of time in culture and maintain the stability of certain phenotypes and functions. Preferably, the cell line is a human embryonic kidney cell line, T cells (e.g., human or mouse T cells), immortalized CD4+ T cell lines (such as the Jurkat cell line), peripheral blood mononuclear cells (PBMCs), stem cells (e.g., mesenchymal stem cells (MSCs) or hematopoietic stem cells), neurons, cardiomyocytes, fibroblasts, Chinese hamster ovary (CHO) cells, or HMEC-1 cells.

[0069] In one implementation, the cells are adherent cells, such as HEK293 cells, HT-1080 cells, or HeLa cells. The term "adherent" implies that the cells grow attached to the surface of the culture vessel.

[0070] In another embodiment, the cells are in suspension in a culture medium, such as Jurkat cells or T cells (e.g., human or mouse T cells; optionally, the T cells are derived from at least one donor).

[0071] In another embodiment, the cells are CD34+ cells. Examples of CD34+ cells may include, but are not limited to, hematopoietic stem cells, early hematopoietic progenitor cells, and B cells.

[0072] In another embodiment, when cells come into contact with the premix in vivo, the cells may be neurons, glial cells, immune cells, inactivated T cells, thymocytes, B cells, myeloid cells, or lymphoid cells.

[0073] As used herein, the term "albumin" includes the meaning of a protein having an amino acid sequence and / or a tertiary structure that is identical to or very similar to that of human serum albumin (particularly SEQ ID NO: 1, which is the mature sequence provided by UniProt P02768), the HSA domain, and may have properties similar to the HSA or related domains. Similar tertiary structures are found in albumins from species other than humans, such as non-human primate albumins (e.g., predicted sequence GenBank XP_517233.2, gorilla albumin, or macaque albumin (e.g., GenBank NP_001182578)), rodent albumins (e.g., hamster albumin (e.g., GenBank A6YF56), guinea pig albumin (e.g., UniProt Q6WDN9-1), mouse albumin (e.g., GenBank AAH49971, mature form with residues 25 to 608 or UniProt P07724-1 version 3) and rat albumin (e.g., GenBank AAH85359 or UniProt P02770-1 version 2)), bovine albumins (e.g., bovine albumin (e.g., UniProt...)). (P02769-1, mature sequence of residues 25 to 607), equine albumin (such as equine 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), canine albumin (e.g., NCBI NP_001003026, mature form of residues 25 to 608), chicken albumin (e.g., UniProt P19121-1 version 2), and porcine albumin (e.g., UniProt P08835-1 version 2), or any of SEQ ID NO: 4 to 19 of WO 2013 / 006675, which is incorporated herein by reference. All of these albumins are included within the scope of this invention. Preferably, the albumin contains the amino acid sequence of mammalian albumin (such as human albumin, bovine albumin, canine albumin, or mouse albumin). Mature forms of albumin (e.g., forms in which all post-translational modifications and / or processing steps have been completed, such as SEQ ID NO: 1) are particularly preferred, and those skilled in the art can identify the mature form using publicly available information (such as protein databases) and / or by using signal peptide identification 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). The albumin formulation used in the methods and compositions of the present invention may contain one or more albumins.

[0074] Some key characteristics of albumin are i) its ability to regulate plasma volume, ii) its long plasma half-life of approximately 19 days ± 5 days, iii) ligand binding, such as binding to endogenous molecules like acidic lipophilic compounds (including bilirubin fatty acids, heme chloride, and thyroxine) (see also Kragh-Hansen et al., 2002, Biol Pharm Bull 25(6):695-704, Table 1, which is incorporated herein by reference), and iv) binding to small molecule organic compounds with acidic or electronegative characteristics (e.g., drugs such as warfarin, diazepam, ibuprofen, and paclitaxel) (see also Kragh-Hansen et al., 2002, Biol Pharm Bull 25(6):695-704, 1, which is incorporated herein by reference). Not all of these characteristics need to be met to characterize a protein or fragment as albumin. If, for example, a fragment does not contain a domain responsible for binding certain ligands or organic compounds, variants of such a fragment are not expected to possess these characteristics.

[0075] The term "albumin" includes fragments and / or variants. Therefore, in one embodiment, albumin is wild-type albumin or a variant thereof. The term "variant thereof" includes a polypeptide derived from parental albumin that contains alterations, namely substitutions, insertions, and / or deletions, at one or more positions. Such alterations may be referred to as "mutations." Substitution includes replacing an amino acid occupying a position with a different amino acid; deletion includes removing an amino acid occupying a position; and insertion includes adding an amino acid (e.g., 1-3 amino acids) near an amino acid occupying a position. For example, altered polypeptides (variants) can be obtained through artificial intervention by modifying the polynucleotide sequence encoding parental albumin.

[0076] In one embodiment, the albumin is a variant of 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 wild-type albumin, such as having 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.

[0077] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRH PYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAE VENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCC KHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0078] Preferably, the albumin has at least 70% sequence identity with mammalian albumin, which is preferably derived 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.

[0079] Mouse albumin sequence (SEQ ID NO: 2):

[0080] EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA

[0081] Canine albumin sequence (SEQ ID NO: 3):

[0082] EAYKSEIAHRYNDLGEEHFRGLVLVAFSQYLQQCPFEDHVKLAKEVTEFAKACAAEESGANCDKSLHTLFGDKLCTVASLRDKYGDMADCCEKQEPDRNECFLAHKDDNPGFPPLVAPEPDALCAAFQDNEQLFLGKYLYEIARRHPYFYAPELLYYAQQYKGVFAECCQAADKAACLGPKIEALREKVLLSSAKERFKCASLQKFGDRAFKAWSVARLSQRFPKADFAEISKVVTDLTKVHKECCHGDLLECADDRADLAKYMCENQDSISTKLKECCDKPVLEKSQCLAEVERDELPGDLPSLAADFVEDKEVCKNYQEAKDVFLGTFLYEYARRHPEYSVSLLLRLAKEYEATLEKCCATDDPPTCYAKVLDEFKPLVDEPQNLVKTNCELFEKLGEYGFQNALLVRYTKKAPQVSTPTLVEVSRKLGKVGTKCCKKPESERMSCAEDFLSVVLNRLCVLHEKTPVSERVTKCCSESLVNRRPCFSGLEVDETYVPKEFNAETFTFHADLCTLPEAEKQVKKQTALVELLKHKPKATDEQLKTVMGDFGAFVEKCCAAENKEGCFSEEGPKLVAAAQAALV

[0083] Bovine albumin sequence (SEQ ID NO: 4):

[0084] DTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPFDEHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELCKVASLRETYGDMADCCEKQEPERNECFLSHKDDSPDLPKLKPDPNTLCDEFKADEKKFWGKYLYEIARRH PYFYAPELLYYANKYNGVFQECCQAEDKGACLLPKIETMREKVLTSSARQRLRCASIQKFGERALKAWSVARLSQKFPKAEFVEVTKLVTDLTKVHKECCHGDLLECADDRADLAKYICDNQDTISSKLKECCDKPLLEKSHCIAE VEKDAIPENLPPLTADFAEDKDVCKNYQEAKDAFLGSFLYEYSRRHPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQNCDQFEKLGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGKVGTRCC TKPESERMPCTEDYLSLILNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADICTLPDTEKQIKKQTALVELLKHKPKATEEQLKTVMENFVAFVDKCCAADDKEACFAVEGPKLVVSTQTALA

[0085] Preferably, the albumin has at least 70% sequence identity with HSA (SEQ ID NO: 1), more preferably 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 with HSA (SEQ ID NO: 1). For example, the preferred albumin has at most 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to wild-type HSA (SEQ ID NO: 1).

[0086] Preferably, the albumin comprises at least 175 consecutive amino acids from albumin having at least 70% sequence identity with HSA (SEQ ID NO: 1), such as 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. The fragment may contain, consist of, or substantially correspond to the following: one or more (e.g., several) domains of albumin such as HSA (SEQ ID NO: 1) or variants 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).

[0087] Typically, variant albumins retain at least one of the major characteristics of the parental albumin or a tertiary structure similar to HSA. For the purposes of this invention, sequence identity between two amino acid sequences can be determined using the Needleman-Wunsch algorithm (Needleman & Wunsch, 1970, J Mol Biol 48(3):443-453), implemented in the Needle program of the EMBOSS software 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. Typical parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 substitution matrix (BLOSUM62 in the EMBOSS version). The Needle output labeled “identity” can be used as an identity percentage, which is the percentage of identical matches between two sequences within the reported alignment region (including any vacancy in length). Alternatively, the Needle output labeled “Longest Identity” (obtained using the -nobrief option) can be used as the identity percentage and can be calculated as follows: (identical residues × 100) / (alignment length - total number of vacancies in the alignment).

[0088] Examples of albumin variants include WO 2011 / 051489, WO 2011 / 124718, WO 2012 / 059486, WO2012 / 150319, WO 2014 / 072481, WO 2013 / 135896, WO 2015 / 036579, WO 2010 / 092135, WO2013 / 075066, WO 2014 / 179657, WO 2009 / 126920, WO 2010 / 059315, WO 2011 / 103076, WO2012 / 112188, WO 2015 / 063611 and WO Those described in 2017 / 029407 (the contents of which are incorporated herein by reference in their entirety, and particularly relate to albumin variants).

[0089] As used herein, the term "parent" or "parental albumin" includes the meaning of albumin from humans or other animals (e.g., mammals). Preferably, other mammalian albumins are albumins derived from clinically relevant animals (such as mice, rats, rabbits, dogs, or guinea pigs). The parent can be a naturally occurring (wild-type) polypeptide or its allele, or a variant as described above.

[0090] As used herein, the term "wild-type albumin" includes albumin having the same amino acid sequence as the major allelic variants of albumin naturally occurring in animals or humans. SEQ ID NO: 1 is an example of wild-type albumin, which is wild-type albumin from Homo sapiens.

[0091] In one embodiment, the albumin is ovalbumin. Ovalbumin (OVA) or albumin refers to the major protein found in egg white. In one embodiment, the egg is a poultry 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:

[0092] MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDED TQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSP

[0093] In one embodiment, the ovalbumin is a variant of 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 wild-type ovalbumin, such as having 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.

[0094] In one embodiment, the ovalbumin has at least 70% sequence identity with wild-type chicken ovalbumin (SEQ ID NO: 5), more preferably 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 with 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 mutation relative to wild-type chicken ovalbumin (SEQ ID NO: 5).

[0095] Preferably, the ovalbumin has 100% identity with wild-type chicken ovalbumin (SEQ ID NO: 5).

[0096] In one embodiment, the albumin is recombinant albumin. In another embodiment, the albumin is serum albumin. In one embodiment, the albumin is recombinant human albumin or human serum albumin. Recombinant human albumin and human serum albumin can have similar abilities to enhance transduction. Recombinant ovalbumin and oval-derived ovalbumin can have similar abilities to enhance transduction.

[0097] In one implementation, albumin is derived from a recombinant source or from serum. "Derived from a recombinant source" includes the meaning that albumin can be derived from recombinant organisms (such as recombinant microorganisms, recombinant plants, or recombinant animals).

[0098] Since some users prefer animal-free components, it is more preferable that albumin is derived from non-animal recombinant sources, such as recombinant microorganisms or recombinant plants. Preferred organisms include prokaryotes, and more preferably, eukaryotes, such as animals, plants, fungi, or yeasts, for example, but not limited to, species in which albumin has been successfully expressed as a recombinant protein, see, for example, the following references incorporated herein by reference:

[0099] - 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))

[0100] - Animals (Barash 1993, Transgenic Research 2, 266-276)

[0101] - Plants (including but not limited to potatoes and tobacco (Sijmons 1990, Biotechnology 8, 217 and Farran 2002, Transgenic Research 11, 337-346) and rice, e.g., Oryzasativa)

[0102] - Mammalian cells, such as CHO and HEK

[0103] - Prokaryotes (Pandjaitab 2000, J. Allergy Clin. Immunol., 105, 279-285), e.g., Escherichia coli (E. coli) (EP73646).

[0104] In one embodiment, the albumin is: a) a yeast-derived albumin, optionally wherein the yeast is a Pichia pastoris, a yeast such as Saccharomyces cerevisiae, Candida, or a Kluyveromyces such as Kluyveromyces lactis or Kluyveromyces marxianus, Hansenula polymorpha, Schizosaccharomyces pombe, Yarrowialipolytica, Arxula adeninivorans, a Candida utilis, or a Zygosaccharomyces such as Zygosaccharomyces bailii; or b) a plant-derived albumin, optionally a rice-derived albumin. Preferably, the albumin is derived from yeast, most preferably from *Saccharomyces cerevisiae* (Celik and Calik (Biotechnology Advances, 2012, 30(5): 1108-1118), Gunduz Ergun et al., “Established and Upcoming Yeast Expression Systems”, “Recombinant Protein Production in Yeast”, 2019, Vol. 1923, pp. 1-74. Edited by Gasser and Mattanovich, published by Humana New York, NY.). Examples of particularly preferred albumins (e.g., recombinant human albumin) include those produced in yeast, particularly *Saccharomyces cerevisiae*, such as the commercially available recombinant yeast-derived albumins known as Recombumin. ® Prime (formerly known as Recombumin) ® ), Recommendbumin ® Elite (formerly known as AlblX) ® ), Recommendbumin ®Alpha (formerly known as Albucult) ® (All of which are from Albumedix Ltd.), or any similar formulation.

[0105] In a preferred embodiment, the premix and / or albumin composition is substantially free of or free of other components, such as antibodies, peptides, proteins, lipids, carbohydrates, small molecule or active pharmaceutical ingredient (API), or biopharmaceuticals (e.g., biopharmaceuticals classified as Class I, II, III, or IV according to the Biopharmaceutical Classification System (BCS) first proposed by Amidon et al. (Pharm Res. 1995, 12(3):413-420)). In other words, the premix preferably comprises albumin and a retroviridae virus, and optionally one or more of a buffer and excipients for the albumin formulation as described herein.

[0106] In one embodiment, the albumin in the premix is ​​present in a concentration of about 1 mg / mL to about 400 mg / mL, such as about 2 mg / mL, 2.1 mg / mL, 2.2 mg / mL, 2.3 mg / mL, 2.4 mg / mL, 2.5 mg / mL, 2.6 mg / mL, 2.7 mg / mL, 2.8 mg / mL, 2.9 mg / mL, 3 mg / mL, 3.1 mg / mL, 3.2 mg / mL, 3.3 mg / mL, 3.4 mg / mL, 3.5 mg / mL, 3.6 mg / mL, 3.7 mg / mL, 3.8 mg / mL, 3.9 mg / mL, 4 mg / mL, 4.5 mg / mL, ... .5mg / mL, 6mg / mL, 6.5mg / mL, 7mg / mL, 7.5mg / mL, 10mg / mL, 15mg / mL, 20mg / mL, 25mg / mL, 30mg / mL, 35mg / mL, 40mg / mL, 45mg / mL, 50mg / mL, 55mg / mL, 60m g / mL, 65mg / mL, 70mg / mL, 75mg / mL, 80mg / mL, 85mg / mL, 90mg / mL, 95mg / mL, 100mg / mL, 105mg / mL, 110mg / mL, 115mg / mL, 120mg / mL, 125mg / mL, 130mg / mL , 135mg / mL, 140mg / mL, 145mg / mL, 150mg / mL, 160mg / mL, 170mg / mL, 180mg / mL, 190mg / mL, 200mg / mL, 210mg / mL, 220mg / mL, 230mg / mL, 235mg / mL, 240mg / mL, 245mg / mL, 250mg / mL to about 100mg / mL, 110mg / mL, 115mg / mL, 120mg / mL, 125mg / mL, 130mg / mL, 135mg / mL, 140mg / mL, 145mg / mL, 150mg / mL, 160mg / mL, 1 70mg / mL, 180mg / mL, 190mg / mL, 200mg / mL, 210mg / mL, 220mg / mL, 230mg / mL, 240mg / mL, 250mg / mL, 260mg / mL, 270mg / mL, 280mg / mL, 290mg / mL, 300mg / m L, 310mg / mL, 320mg / mL, 330mg / mL, 340mg / mL, 350mg / mL, 355mg / mL, 360mg / mL, 365mg / mL, 370mg / mL, 375mg / mL, 380mg / mL, 385mg / mL, 390mg / mL, 392.The albumin in the premix is ​​present at concentrations of 5 mg / mL, 395 mg / mL, 397.5 mg / mL, 398 mg / mL, 399 mg / mL, and 400 mg / mL. Preferably, the albumin in the premix has a concentration of about 2 mg / mL to about 400 mg / mL, such as about 2.5 mg / mL to about 400 mg / mL, about 3 mg / mL to about 400 mg / mL, about 3.5 mg / mL to about 400 mg / mL, about 4 mg / mL to about 400 mg / mL, about 4.5 mg / mL to about 400 mg / mL, about 4.75 mg / mL to about 400 mg / mL, about 10 mg / mL to about 250 mg / mL, about 25 mg / mL to about 250 mg / mL, about 95 mg / mL to about 210 mg / mL, about 10 mg / mL to about 100 mg / mL, more preferably about 25 mg / mL to about 100 mg / mL, and most preferably about 50 mg / mL.

[0107] Typically, the premix is ​​brought into contact with cells located in the wells of a reservoir or container, such as a plate (e.g., a multi-well plate). It should be understood that once the premix is ​​added to the cells, albumin can be diluted, and therefore the albumin concentration in the composition containing the premix and cells (e.g., the composition in the wells of a multi-well plate) may be low. For example, the albumin concentration in the composition containing the premix and cells (e.g., the composition in the wells of a multi-well plate) can be diluted up to approximately 100-fold compared to the albumin concentration in the premix before it is added to the cells. Therefore, in one embodiment, the composition comprising the premix and cells (e.g., the composition in the wells of a multi-well plate) contains concentrations from about 0.01 mg / mL to about 4 mg / mL, such as about 0.01 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.075 mg / mL, 0.100 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, or 0.65 mg / mL to about 0.02 mg / mL, 0.025 mg / mL. , 0.05mg / mL, 0.075mg / mL, 0.08mg / mL, 0.085mg / mL, 0.09mg / mL, 0.095mg / mL, 0.1mg / mL, 0.25mg / mL, 0.5mg / mL, 0.75mg / mL, 0.8mg / mL, 0.85mg / mL, 0. Albumin at 9 mg / mL, 0.95 mg / mL, 0.975 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 3.6 mg / mL, 3.7 mg / mL, 3.8 mg / mL, 3.9 mg / mL, or 4 mg / mL. Preferably, the albumin concentration in the composition comprising the premix and cells (e.g., the composition in the wells of a multi-well plate) is about 0.02 mg / mL to about 4 mg / mL, about 0.025 mg / mL to about 4 mg / mL, about 0.03 mg / mL to about 4 mg / mL, about 0.035 mg / mL to about 4 mg / mL, about 0.04 mg / mL to about 4 mg / mL, about 0.045 mg / mL to about 4 mg / mL, about 0.0475 mg / mL to about 4 mg / mL, about 0.1 mg / mL to about 2.5 mg / mL, about 0.25 mg / mL to about 2.5 mg / mL, about 0.95 mg / mL to about 2.1 mg / mL, about 0.1 mg / mL to about 1 mg / mL, more preferably about 0.25 mg / mL to about 1 mg / mL, and most preferably about 0.5 mg / mL.

[0108] It should be understood that premixes can be isolated from the body or outside the body, or in contact with cells within the body.

[0109] In one implementation, a retroviridae virus is a virus from the subfamily Orthorevirusinae or the foamy retrovirusinae, or any virus derived therefrom. Viruses from the subfamily Orthorevirusinae are defined by their ability to reverse transcribe their RNA genome into DNA that integrates into the cellular genome during the virus's intracellular life cycle. Viruses from the subfamily Foamy retrovirusinae contain double-stranded DNA.

[0110] In one embodiment, the positronovirinae virus is a lentivirus or any virus derived therefrom. In another embodiment, the positronovirinae virus is any one of alpha retrovirus, beta retrovirus, delta retrovirus, ε retrovirus, gamma retrovirus, or any virus derived therefrom.

[0111] In one implementation, the gamma retrovirus is any of murine leukemia virus, Abelson murine leukemia virus, Friend virus, feline leukemia virus, koala retrovirus (KoRV), heterophilic murine leukemia virus-associated virus, or any virus derived therefrom.

[0112] In one implementation, the foam retrovirinae virus is any one of bovine foam virus, equine foam virus, feline foam virus, prosimian foam virus, or simian foam virus, or any virus derived therefrom.

[0113] In one implementation, the lentivirus is any one of human immunodeficiency virus (HIV) such as HIV-1 or HIV-2, simian immunodeficiency virus (SIV), mouse lentivirus, equine infectious encephalitis virus, equine infectious anemia virus (EIAV), caprine arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BIV), or feline immunodeficiency virus (FIV), or any virus derived therefrom.

[0114] In one implementation, the retroviridae virus is any of a viral particle, a viral vector (such as a retroviridae virus vector), or a vector virus.

[0115] As used herein, the term Retroviridae viruses include viruses, viral particles, viral fragments, virus-like particles (VLPs), viral vectors, or vector viruses. As used herein, the terms "viral particle" and "virus-like particle" refer to a non-replicating viral capsid derived from any of the viruses discussed above. Viral particles and VLPs are typically composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsids, shells, capsids, surface, and / or envelope proteins, or particle-forming polypeptides derived from these proteins. Furthermore, they may or may not contain additional genetic material. The term "viral particle" refers to the whole virus that is infectious unless attenuated or inactivated. VLPs are non-infectious particles. As used herein, the term "viral fragment" refers to a portion or part of a virus or viral particle, preferably non-replicating. As used herein, the terms "viral vector" and "vector virus" refer to a vector (e.g., a modified virus) used to deliver genetic material into cells. Viral vectors utilize mechanisms that viruses have evolved to efficiently transport their genome into the cells they infect. Viral vectors and vector viruses are commonly used in basic research, gene therapy, and vaccine development.

[0116] In the context of this invention, lentiviral vector refers to a non-replicating vector intended for transduction of cells with a transgenic lentiviral RNA or DNA sequence comprising a cis-acting lentiviral RNA or DNA sequence, and requiring trans-provided lentiviral proteins (e.g., one or more or all of Gag, Pol, and / or Env). Lentiviral vectors may lack expression of one or more or all of the functional Gag, Pol, and Env proteins. Lentiviral vectors may be present in the form of RNA or DNA molecules, depending on the stage of production or development of the retroviral vector. For the avoidance of doubt, it should be understood that transduction (the process of introducing exogenous nucleic acid into another cell via a viral vector) is different from transfection (the process of introducing nucleic acid into a cell by a non-viral method).

[0117] Lentiviral vectors can be in the form of recombinant DNA molecules, such as plasmids. Lentiviral vectors can also be in the form of lentiviral vector particles, such as RNA molecules within a complex of lentivirus and other proteins. Typically, lentiviral vector particles corresponding to modified or recombinant lentiviral particles contain a genome consisting of two copies of single-stranded RNA. These RNA sequences can be obtained by transcription from a double-stranded DNA sequence inserted into the cell genome (proviral vector DNA), or from transient expression of plasmid DNA (plasmid vector DNA) in transformed cells.

[0118] Typically, lentiviral vector particles possess integration capability. Therefore, they contain functional integrase proteins. Non-integrating vector particles have one or more mutations that eliminate most or all of the integration capability of lentiviral vector particles. For example, non-integrating vector particles may contain mutations in the integrase encoded by the lentiviral pol gene, which result in reduced integration capability. In contrast, integrating vector particles contain functional integrase proteins and do not contain any mutations that eliminate most or all of the integration capability of lentiviral vector particles.

[0119] Lentiviral vectors are derived from lentiviruses, particularly 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 that can be used to obtain therapeutic effects.

[0120] These vectors are based on the separation of cis and trans-acting sequences. To generate replication-defective vectors, trans-acting sequences (e.g., gag, pol, tat, rev, and env genes) can be deleted and replaced with expression cassettes encoding transgenes.

[0121] In one implementation scheme, the retroviridae virus is a live retroviridae virus, an attenuated retroviridae virus, or a live attenuated retroviridae virus.

[0122] In one embodiment, the retroviral virus is the form of a vaccine, preferably wherein the vaccine is a virus-based vaccine. Lentiviral vectors have a high potential to transduce dendritic cells in vivo. Within these cells, which most effectively activate naïve T cells, the lentiviral vector induces endogenous expression of transgenic antigens that directly enter the antigen presentation pathway without external antigen capture or cross-presentation. For example, a vaccine based on a retroviral virus can provide protection against: (i) viruses, such as those from the Flaviviridae family (e.g., West Nile virus (WNV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), dengue virus (DENV)), SARS-CoV-2; bacterial infections (e.g., tuberculosis); or parasitic infections (e.g., malaria).

[0123] In one embodiment, the retroviridae virus in the premix is ​​present in a multiple of infection (MOI) range of 0.1-100, such as 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 virus in the premix is ​​present in an MOI range of 1-10. Most preferably, the retroviridae virus in the premix is ​​present in an MOI of 5.

[0124] The term "multiple of infection" or "MOI" refers to the ratio of the number of viral particles to the number of target cells present. Generally, as the MOI increases, the percentage of cells infected with at least one viral particle also increases. Those skilled in the art can determine the MOI, such as... The optimal MOI can be determined experimentally by those skilled in the art.

[0125] In one embodiment, the premix also comprises a buffer solution. For example, one or both of retroviridae virus and albumin may be provided in the buffer solution and mixed together to form a premix comprising the buffer solution. Alternatively, both retroviridae virus and albumin may be added to the buffer solution to form a premix comprising retroviridae virus, albumin, and buffer solution.

[0126] In one embodiment, the buffer solution has a buffering capacity of about pH 6.0 to about pH 8.0, such as 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.

[0127] In one implementation, the buffer is phosphate-buffered saline (PBS), Hanks' buffered salt solution, Earle's balanced salt solution, Tyrode's solution, MOPS buffer, HEPES buffer, or any isotonic solution for injection (such as Plasma-Lyte). ® For example, Plasma-Lyte ® Either 148 or Ringer lactate. Preferably, the buffer is PBS.

[0128] In one embodiment, the premix comprises 1 mmol to 250 mmol (mM) of buffer solution. The buffer solution concentration can be about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM or 100 mM to about 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 150 mM, 200 mM or 250 mM. The preferred buffer concentration is from about 10 mM to about 200 mM, particularly about 150 mM, especially where the buffer is PBS.

[0129] Albumin added to retroviridae viruses to form premixes can be provided in the form of albumin formulations. Formulations are typically liquid albumin formulations, but it should be understood that formulations can be dried formulations, such as freeze-dried formulations.

[0130] In one embodiment, the albumin used to form the premix is ​​provided in the form of a formulation, and a suitable formulation is any one of the following: (a) 13 mM to 16 mM, preferably about 14.5 mM, sodium; 2.9 mM to 3.5 mM, preferably about 3.2 mM, caprylate; 1 mg / L to 2 mg / L, preferably 1 mg / L to 1.5 mg / L, polysorbate 80; and 19 mg / L to 21 mg / mL, preferably about 20 mg / mL, albumin (w / v), wherein the pH is 6.7 to 7.3, preferably about 7; (b) Sodium at 12 mM to 16 mM, preferably about 14.5 mM; octanoate at 0.4 mM to 1.2 mM, preferably about 0.8 mM; polysorbate 80 at 0 mg / L to 5 mg / L, preferably about 2.5 mg / L to 4.5 mg / L; and albumin (w / v) at 9.5 mg / mL to 10.5 mg / mL, preferably about 10 mg / mL, wherein the pH is 6.4 to 7.4, preferably about 7; (c) (d) Sodium 12mM to 16mM, preferably about 14.5mM; octanoate 0.8mM to 2.4mM, preferably about 1.6mM; polysorbate 80 0mg / L to 10mg / L, preferably 5mg / L to 7mg / L; albumin (w / v) 19mg / mL to 21mg / mL, preferably about 20mg / mL, wherein the pH is 6.4 to 7.4, preferably about 7; (d) Sodium 20mM to 30mM, preferably 23mM to 26mM; octanoate 0mM to 0.3mM, preferably 0mM to 0.1mM; albumin (w / v) 9.5mg / mL to 10.5mg / mL, preferably about 10mg / mL, wherein the pH is 6 to 7, preferably about 6.5.

[0131] In another embodiment, a suitable formulation is any one of the following groups: (a) 130 mM to 160 mM, preferably about 145 mM, sodium; 29 mM to 35 mM, preferably about 32 mM, caprylate; 10 mg / L to 20 mg / L, preferably 10 mg / L to 15 mg / L, polysorbate 80; and 190 mg / mL to 210 mg / mL, preferably about 200 mg / mL, albumin (w / v), wherein the pH is 6.7 to 7.3, preferably about 7; (b) (c) Sodium 120mM to 160mM, preferably about 145mM; octanoate 4mM to 12mM, preferably about 8mM; polysorbate 80 0 mg / L to 50 mg / L, preferably 25 mg / L to 45 mg / L; albumin 95 mg / mL to 105 mg / mL, preferably about 100 mg / mL (w / v), wherein the pH is 6.4 to 7.4, preferably about 7; (d) Sodium 120mM to 160mM, preferably about 145mM; octanoate 80 8mM 8M, preferably about 16mM; polysorbate 80 0 mg / L to 100 mg / L, preferably 50 mg / L to 70 mg / L; albumin 190 mg / mL to 210 mg / mL, preferably about 200 mg / mL (w / v), wherein the pH is 6.4 to 7.4, preferably about 7; Sodium at 200 mM to 300 mM, preferably 230 mM to 260 mM, octanoate at 0 mM to 3 mM, preferably 0 mM to 1 mM, and albumin at 95 mg / mL to 105 mg / mL, preferably about 100 mg / mL, wherein the pH is 6 to 7, preferably about 6.5.

[0132] In another embodiment, a suitable formulation is any one of the following groups: (a) 6.5 mM to 8 mM, preferably about 7.25 mM, sodium; 1.45 mM to 1.75 mM, preferably about 1.6 mM, caprylate; 0.5 mg / L to 1 mg / L, preferably 0.5 mg / L to 0.75 mg / L, polysorbate 80; and 9.5 mg / mL to 10.5 mg / mL, preferably about 10 mg / mL, albumin (w / v), wherein the pH is 6.7 to 7.3, preferably about 7; (b) Sodium 6 mM to 8 mM, preferably about 7.25 mM; octanoate 0.2 mM to 0.6 mM, preferably about 0.4 mM; polysorbate 80 0 mg / L to 2.5 mg / L, preferably 1.25 mg / L to 2.25 mg / L; albumin (w / v) 4.75 mg / mL to 5.25 mg / mL, preferably about 5 mg / mL, wherein the pH is 6.4 to 7.4, preferably about 7; (c) 6 mM to 8 mM, preferably about 7.25 mM sodium, 0.4 mM to 1.2 mM, preferably about 0.8 mM caprylate, 0 mg / L to 5 mg / L, preferably about 2.5 mg / L to 3.5 mg / L polysorbate 80, 9.5 mg / mL to 10.5 mg / mL, preferably about 10 mg / mL albumin (w / v), wherein the pH is 6.4 to 7.4, preferably 7; (d) 10 mM to 15 mM, preferably 11.5 mM to 13 mM sodium, 0 mM to 0.15 mM, preferably 0 mM to 0.05 mM caprylate, 4.75 mg / mL to 5.25 mg / mL, preferably about 5 mg / mL albumin (w / v), wherein the pH is 6 to 7, preferably about 6.5.

[0133] In one embodiment, the albumin formulation contains albumin at a concentration of 2 mg / mL to 400 mg / mL, such as 2.5 mg / mL to 400 mg / mL, 4.5 mg / mL to 400 mg / mL or 4.75 mg / mL to 400 mg / mL, preferably 10 mg / mL to 250 mg / mL mM, such as 95 mg / mL to 210 mg / mL, more preferably 10 mg / mL to 100 mg / mL, and most preferably about 10 mg / mL to about 20 mg / mL.

[0134] In one embodiment, the albumin formulation contains sodium at a concentration of 0.4 mM to 300 mM or 1 mM to 300 mM (e.g., 50 mM-300 mM or 120 mM-300 mM), preferably 4 mM to 25 mM or 10 mM to 50 mM, more preferably 5 mM to 14 mM or 12 mM to 30 mM, and most preferably about 11 mM or about 25 mM.

[0135] In one embodiment, the albumin formulation contains caprylate at a concentration of 0 mM to 35 mM or 0 mM to 16 mM, preferably 0 mM to 5 mM or 0 mM to 2.5 mM, more preferably 0 mM to 3.5 mM or 0 mM to 1.6 mM, and most preferably about 0.1 mM or about 0.04 mM.

[0136] In one embodiment, the albumin formulation contains polysorbate 80 at a concentration of 0 mg / mL to 100 mg / mL or 0 mg / mL to 45 mg / mL, preferably 0 mg / mL to 10 mg / mL or 0 mg / mL to 4.5 mg / mL, more preferably 0 mg / mL to 5 mg / mL or 0 mg / mL to 2.5 mg / mL, and most preferably about 2.5 mg / mL or about 1.1 mg / mL.

[0137] In one embodiment, the premix comprises 2 mg / mL to 400 mg / mL or 2.5 mg / mL to 400 mg / mL albumin (w / v), such as 4.5 mg / mL to 400 mg / mL or 4.75 mg / mL to 400 mg / mL albumin (w / v), preferably 10 mg / mL to 250 mg / mL, such as 95 mg / mL to 210 mg / mL, more preferably 10 mg / mL to 100 mg / mL, and most preferably about 10 mg / mL to about 20 mg / mL albumin (w / v).

[0138] In one embodiment, the premix contains 0.4 mM to 300 mM or 1 mM to 300 mM, preferably 4 mM to 50 mM or 10 mM to 50 mM, more preferably 5 mM to 30 mM or 12 mM to 30 mM, and most preferably about 11 mM or about 25 mM of sodium.

[0139] In another embodiment, the premix contains 0.7 mM to 270 mM (e.g., 84 mM-270 mM), preferably 7 mM to 45 mM, more preferably 8.4 mM to 27 mM, and most preferably about 22.5 mM or about 17.5 mM of sodium.

[0140] In one embodiment, the premix comprises 0 mM to 35 mM or 0 mM to 16 mM, preferably 0 mM to 5 mM or 0 mM to 2.5 mM, more preferably 0 mM to 3.5 mM or 0 mM to 1.6 mM, and most preferably about 0.1 mM or about 0.04 mM of octanoate.

[0141] In another embodiment, the premix comprises 0 mM to 24.5 mM or 0 mM to 31.5 mM, preferably 0 mM to 3.5 mM or 0 mM to 4.5 mM, more preferably 0 mM to 2.5 mM or 0 mM to 3.2 mM, and most preferably about 0.07 mM or about 0.09 mM of octanoate.

[0142] In one embodiment, the premix comprises 0 mg / mL to 100 mg / mL or 0 mg / mL to 45 mg / mL, preferably 0 mg / mL to 10 mg / mL or 0 mg / mL to 4.5 mg / mL, more preferably 0 mg / mL to 5 mg / mL or 0 mg / mL to 0.25 mg / mL, and most preferably about 2.5 mg / mL or about 1.1 mg / mL of polysorbate 30 or polysorbate 80, preferably polysorbate 80.

[0143] In another embodiment, the premix comprises 0 mg / mL to 70 mg / mL or 0 mg / mL to 90 mg / mL, preferably 0 mg / mL to 7 mg / mL or 0 mg / mL to 9 mg / mL, more preferably about 0 mg / mL to 3.5 mg / mL or about 0 mg / mL 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.

[0144] In one embodiment, the premix comprises a combination of albumin (w / v) at a concentration of 2 mg / mL to 400 mg / mL or 2.5 mg / mL to 400 mg / mL, such as albumin (w / v) at a concentration of 4.5 mg / mL to 400 mg / mL or 4.75 mg / mL to 400 mg / mL, and sodium at a concentration of 0.4 mM to 300 mM, 1 mM to 270 mM, or 1 mM to 300 mM.

[0145] In one embodiment, the premix comprises a combination of albumin (w / v) at a concentration of 2 mg / mL to 400 mg / mL or 2.5 mg / mL to 400 mg / mL, such as albumin (w / v) at a concentration of 4.5 mg / mL to 400 mg / mL or 4.75 mg / mL to 400 mg / mL, with octanoate at a concentration of 0 mM to 35 mM, 0 mM to 16 mM, or 0 mM to 31.5 mM.

[0146] In one embodiment, the premix comprises 2 mg / mL to 400 mg / mL or 2.5 mg / mL to 400 mg / mL albumin (w / v), such as 4.5 mg / mL to 400 mg / mL or 4.75 mg / mL to 400 mg / mL albumin (w / v), and 0 mg / mL to 100 mg / mL or 0 mg / mL to 45 mg / mL or 0 mg / mL to 90 mg / mL polysorbate 30 or polysorbate 80, preferably polysorbate 80.

[0147] In one embodiment, the premix comprises 2 mg / mL to 400 mg / mL or 2.5 mg / mL to 400 mg / mL albumin (w / v), such as 4.5 mg / mL to 400 mg / mL or 4.75 mg / mL to 400 mg / mL albumin (w / v), combined with 0.4 mM to 300 mM, 1 mM to 270 mM or 1 mM to 300 mM sodium and 0 mM to 16 mM, 0 mM to 31.5 mM or 0 mM to 35 mM octanoate.

[0148] In one embodiment, the premix comprises 2 mg / mL to 400 mg / mL or 2.5 mg / mL to 400 mg / mL albumin (w / v), such as 4.5 mg / mL to 400 mg / mL or 4.75 mg / mL to 400 mg / mL albumin (w / v), with 0.4 mM to 300 mM, 1 mM to 270 mM or 1 mM to 300 mM sodium and 0 mg / mL to 45 mg / mL, 0 mg / mL to 90 mg / mL or 0 mg / mL to 100 mg / mL polysorbate 30 or polysorbate 80, preferably polysorbate 80.

[0149] In one embodiment, the premix comprises 2 mg / mL to 400 mg / mL or 2.5 mg / mL to 400 mg / mL albumin (w / v), such as 4.5 mg / mL to 400 mg / mL or 4.75 mg / mL to 400 mg / mL albumin (w / v), with 0 mM to 16 mM, 0 mM to 31.5 mM or 0 mM to 35 mM caprylate and 0 mg / mL to 45 mg / mL, 0 mg / mL to 90 mg / mL or 0 mg / mL to 100 mg / mL polysorbate 30 or polysorbate 80, preferably polysorbate 80.

[0150] In one embodiment, the premix and / or albumin composition is substantially free of or contains no fatty acids.

[0151] In one embodiment, the enhancement of transduction is greater than or equal to the enhancement of transduction provided by using a known transduction enhancer (e.g., replacing albumin in the premix with a known transduction enhancer, or adding a known transduction enhancer to the cells without forming a premix). Transduction enhancers are known in the art and include enhancers such as Lentiboost. ® (Sirion Biotech), polygluconate (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 sulfate or combinations thereof. Therefore, in one embodiment, the enhancement of transduction is greater than or equal to that achieved by Lentiboost. ® (Sirion Biotech), polygluconate (Merck Life Sciences), poloxamer 338 (also known as poloxamer Synperonic) ® F108), Lenti-X ™ Accelerator (Takara Bio Inc.), RetroNectin ® (Takara Bio Inc), Vectofusion ® Enhancement of transduction provided by any or more of the following: (Miltenyi Biotec Ltd) or protamine sulfate or combinations thereof.

[0152] The enhancement of transduction greater than that provided by using a known transduction enhancer includes the following meanings: the transduction is increased by at least 1% compared to the transduction provided 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%, or 200%. Preferably, the transduction is increased by at least 5% compared to the transduction provided by using a known transduction enhancer.

[0153] In one implementation, the cells are treated with one or more known transduction enhancers, such as Lentiboost. ®(Sirion Biotech), polygluconate (Merck Life Sciences), poloxamer 338 (also known as poloxamer Synperonic) ® F108), Lenti-X ™ Accelerator (Takara Bio Inc.), RetroNectin ® (Takara BioInc), Vectofusion ® Further contact with either (Miltenyi Biotec Ltd) or protamine sulfate or combinations thereof. Preferably, one or more transduction enhancers are added to the cells before the premix is ​​added to the cells. However, it should be understood that one or more transduction enhancers may be formed as part of the premix and added to the cells once formed.

[0154] It should be understood that when cells are further contacted with one or more known transduction enhancers, the enhancement of transduction can be greater than that provided when cells are contacted only with the premix without one or more known transduction enhancers and / or when cells are contacted only with one or more known transduction enhancers without the premix. Transduction can increase by at least 1%, more preferably 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%, compared to that provided when cells are contacted only with the premix without one or more known transduction enhancers and / or when cells are contacted only with one or more known transduction enhancers without the premix. Preferably, the increase in transduction is at least 5% compared to that provided when cells are contacted only with the premix without one or more known transduction enhancers and / or when cells are contacted only with one or more known transduction enhancers without the premix. For example, preferably, the transduction is increased by at least 5% compared to the transduction provided when cells are only contacted with the premix without one or more known transduction enhancers, and the increase is at least 5% when cells are only contacted with one or more known transduction enhancers without the premix. Most preferably, when cells are contacted with both the premix and one or more known transduction enhancers, the enhancement of transduction is greater than the sum of the enhancements of transduction caused by the premix and the one or more known transduction enhancers individually. In other words, the addition of the premix and one or more known transduction enhancers can produce a synergistic effect on transduction enhancement.

[0155] Lentiboost ® It is a commercial transduction enhancer and has a proprietary formulation containing poloxamer 338 and optional polybrene.

[0156] In one embodiment, Lentiboost is included in a composition comprising a premix and cells. ® The concentration is from about 0.01 mg / mL to about 10 mg / mL, such as about 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL or 6 mg / mL to about 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 9.5 mg / mL, 9.8 mg / mL, 9.9 mg / mL or 10 mg / mL. Preferably, Lentiboost is present in compositions comprising the premix and cells. ® The concentration is from about 0.1 mg / mL to about 2 mg / mL. Most preferably, Lentiboost is present in the composition comprising the premix and cells. ® The concentration is 1 mg / mL.

[0157] In one implementation, Lentiboost in the premix ® The concentration is from about 0.1 mg / mL to about 1000 mg / mL, such as about 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 50 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL, 500 mg / mL, or 600 mg / mL to about 200 mg / mL, 300 mg / mL, 400 mg / mL, 500 mg / mL, 600 mg / mL, 700 mg / mL, 800 mg / mL, 900 mg / mL, 950 mg / mL, 980 mg / mL, 990 mg / mL, or 1000 mg / mL. Preferably, Lentiboost in the premix... ® The concentration is from about 10 mg / mL to about 200 mg / mL, such as from about 10 mg / mL to about 100 mg / mL. Most preferably, the premix contains Lentiboost. ® The concentration is 0.1 mg / mL to 5 mg / mL, such as about 0.2 mg / mL, 0.3 mg / mL or 0.4 mg / mL.

[0158] Poloxamer 388 or Poloxamer Synperonic ®F108 is defined as a "large nonionic amphiphilic molecule" according to the following formula, as in WO 2013 / 127964:

[0159]

[0160] In one embodiment, poloxamer 338 or poloxamer Synperonic is included in the composition comprising the premix and cells (e.g., in the wells of a multi-well plate). ® The concentration of F108 is about 0.5 µg / mL to about 50 µg / mL, such as about 0.5 µg / mL, 0.55 µg / mL, 0.6 µg / mL, 0.65 µg / mL, 0.75 µg / mL, 1 µg / mL, 2 µg / mL. mL, 3µg / mL, 4µg / mL, 5µg / mL, 6µg / mL, 7µg / mL, 8µg / mL, 9µg / mL, 10µg / mL, 11µg / mL, 12µg / mL, 13µg / mL, 14µg / m L or 15µg / mL to approximately 10µg / mL, 11µg / mL, 12µg / mL, 13µg / mL, 14µg / mL, 15µg / mL, 16µg / mL, 17µg / mL, 18µg / mL, 19µg / m L, 20µg / mL, 25µg / mL, 30µg / mL, 35µg / mL, 40µg / mL, 45µg / mL, 46µg / mL, 47µg / mL, 48µg / mL, 49µg / mL or 50µg / mL.

[0161] In one embodiment, the premix contains poloxamer 338 or poloxamer Synperonic ®The concentration of F108 is from about 50 µg / mL to about 5000 µg / mL, such as about 50 µg / mL, 55 µg / mL, 60 µg / mL, 65 µg / mL, 70 µg / mL, 75 µg / mL, 100 µg / mL, 200 µg / mL, 300 µg / mL, 400 µg / mL, 500 µg / mL, 600 µg / mL, 700 µg / mL, 800 µg / mL, 900 µg / mL, 1000 µg / mL, 1100 µg / mL, 1200 µg / mL, 1300 µg / mL, 1400 µg / mL, or 1500 µg / mL. g / mL to approximately 1000µg / mL, 1100µg / mL, 1200µg / mL, 1300µg / mL, 1400µg / mL, 1500µg / mL, 1600µg / mL, 1700µg / mL, 1800µg / mL, 1900µg / mL, 20 00µg / mL, 2500µg / mL, 3000µg / mL, 3500µg / mL, 4000µg / mL, 4500µg / mL, 4600µg / mL, 4700µg / mL, 4800µg / mL, 4900µg / mL or 5000µg / mL.

[0162] Polyamide is a polycationic 1,5-dimethyl-1,5-diaza-undecanyl-polymethyl bromide as defined in WO 2013 / 127964:

[0163]

[0164] Polybrene reduces the electrostatic repulsion between viruses and cell membranes. The optimal final concentration of polybrene can be determined by those skilled in the art. Excessive exposure to polybrene (such as for more than 24 hours) may be toxic to cells.

[0165] In one embodiment, the concentration of polybrene in the composition comprising the premix and cells (e.g., in the wells of a multi-well plate) is from about 1 μg / mL to about 20 μg / mL, such as about 1.0 μg / mL, 1.5 μg / mL, 2.0 μg / mL, 2.5 μg / mL, 3.0 μg / mL, 3.5 μg / mL, 4.0 μg / mL, 4.5 μg / mL, 5.0 μg / mL, 5.5 μg / mL, 6.0 μg / mL, 6.5 μg / mL, 7.0 μg / mL, 7.5 μg / mL, 8 μg / mL. The concentrations are 0 μg / mL, 8.5 μg / mL, 9.0 μg / mL, 9.5 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, or 14 μg / mL to about 9 μg / mL, 9.5 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, 19.5 μg / mL, or 20 μg / mL. Preferably, the concentration of polybrene in the composition comprising the premix and cells is about 2 μg / mL to about 12 μg / mL. Most preferably, the concentration of polybrene in the composition comprising the premix and cells is about 8 μg / mL.

[0166] In one embodiment, the concentration of polybrene in the premix is ​​from about 100 μg / mL to about 2 mg / mL, such as about 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, 500 μg / mL, 550 μg / mL, 600 μg / mL, 650 μg / mL, 700 μg / mL, 750 μg / mL, 800 μg / mL, 850 μg / mL. The concentrations are 900 μg / mL, 950 μg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, or 1.4 mg / mL to about 900 μg / mL, 950 μg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 1.95 mg / mL, or 2 mg / mL. Preferably, the concentration of polybrene in the premix is ​​about 200 μg / mL to about 1.2 mg / mL. Most preferably, the concentration of polybrene in the premix is ​​about 800 μg / mL.

[0167] When cells are further exposed to one or more transduction enhancers, the enhancement of transduction may be even greater than the enhancement already provided by albumin in the premix (i.e., without the additional step of further exposing cells to transduction enhancers). In one embodiment, the enhancement of transduction is at least 1.01-fold, 1.02-fold, 1.03-fold, 1.04-fold, 1.05-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4.0-fold, 4.1-fold, 4.2-fold, 4.3-fold, 4.4-fold, 4.5-fold, 4.6-fold, 4.7-fold, 4 ... 8 times, 4.9 times, 5.0 times, 5.1 times, 5.2 times, 5.3 times, 5.4 times, 5.5 times, 5.6 times, 5.7 times, 5.8 times, 5.9 times, 6.0 times, 6.1 times, 6.2 times, 6.3 times, 6.4 times, 6.5 times, 6.6 times, 6.7 times, 6.8 times, 6.9 times, 7.0 times, 7.1 times, 7.2 times, 7.3 times, 7.4 times 7.5 times, 7.6 times, 7.7 times, 7.8 times, 7.9 times, 8.0 times, 8.1 times, 8.2 times, 8.3 times, 8.4 times, 8.5 times, 8.6 times, 8.7 times, 8.8 times, 8.9 times, 9.0 times, 9.1 times, 9.2 times, 9.3 times, 9.4 times, 9.5 times, 9.6 times, 9.7 times, 9.8 times, 9.9 times, or at least 10 times. For example, an enhancement of "at least 1.1 times" means an increase in transduction of at least 10%.

[0168] In one implementation, the use of albumin and one or more transduction enhancers can have a synergistic effect on transduction enhancement. The term "synergistic effect" implies that the increase in transduction is significantly greater than the sum of the effects of the albumin and transduction enhancer combination.

[0169] As shown by the inventors in Example 1, it was found that the effect of albumin on lentivirus varies depending on how long the premix is ​​incubated before contacting the cells. For example, if the lentivirus is stored in albumin before being added to the cells, the albumin both stabilizes the virus and enhances transduction, while if albumin is mixed with the lentivirus and then the premix is ​​immediately transferred to the cells, the lentivirus only enhances transduction. Therefore, in one embodiment, particularly where the premix is ​​incubated for a certain period of time before contacting the cells in step (ii), the method of the present invention also increases the stability of retroviridae viruses (e.g., retroviruses or lentiviruses).

[0170] Therefore, the present invention also provides a method for (a) enhancing the transduction of retroviridae viruses into cells and (b) increasing the stability of retroviridae viruses, the method comprising:

[0171] (i) Combining retroviridae viruses with albumin to form a premix; and

[0172] (ii) Contact the cells with the premix formed in step (i).

[0173] The term "stability" of retroviridae viruses encompasses the meaning of maintaining their integrity and functionality. The stability of retroviridae viruses can be assessed using any suitable method known in the art. For example, low stability can affect the quality and efficacy of retroviridae viruses. This is characterized by a significant decrease in the titer of the retroviridae virus over time. Existing techniques for increasing viral stability include maintaining the formulation at very low temperatures (e.g., -90°C to -70°C). However, after multiple freeze-thaw cycles or after a very short thawing period, viral viability is lost. This can lead to the waste of non-negligible amounts of expensive pharmaceutical or vaccine formulations.

[0174] The phrase "increased stability of retroviridae viruses" implies an increase in viral stability compared to the stability of retroviridae viruses in the absence of albumin. In other words, when assessing viral stability under specific conditions, exposing the virus to albumin should increase viral stability under the same specific conditions. The selected conditions can be any suitable “stress conditions” known to affect the stability of the virus, such as 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 days, 3 days, 4 days, 5 days, or 6 days, at least 1 week, or at least 2 weeks, or at least 1 month or 2 months)) or at 2°C to 8°C, such as about 2°C, 3°C, 4°C, 5°C, 6°C, or 7°C to about 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C, such as about 4°C) for at least 2 months.

[0175] Assessing whether the stability of a retroviridae virus has increased can be performed using any suitable technique known in the art and as described in Example 1. For example, viral aggregation can be assessed, for instance, by measuring the hydrodynamic radius of the preparation containing the virus. Dynamic light scattering (DLS) can be used. Stability can be determined by measuring the virus's ability to bind to a binding chaperone (e.g., a monoclonal antibody) or infect cells.

[0176] Retroviridae viruses are commonly used in therapy, so enhancing viral transduction efficiency into cells would be particularly beneficial for improving the efficacy of such therapy. Therefore, in a second aspect, the present invention provides a method for improving the efficacy of a retroviridae virus-based therapy, the method comprising combining the retroviridae virus with albumin to form a premix; and administering the premix to a subject.

[0177] Preferred options for retroviral viruses, albumins, and premixes include those described above with respect to the first aspect of the invention.

[0178] The term "retroviral therapy" encompasses the use of retroviral viruses to treat and / or prevent diseases or symptoms from which therapies would benefit. For example, a retroviral virus-based therapy could be a gene-based therapy, where a retroviral virus is used as a vector to transfer a specific gene or a portion thereof into cells, thereby preventing and / or treating a disease or symptom. Examples of gene therapies based on retroviral viruses include the use of lentiviruses to deliver β-globulin, γ-chain (γc), and tumor-specific T-cell receptors (TCRs). Other examples include ADA cDNA (e.g., for treating ADA-SCID), IL2RG cDNA (e.g., for treating SCID), CYBB cDNA (for treating XL CGD), and CD4 binding agents. Further examples of gene therapies based on retroviral viruses include the use of retroviruses to deliver drug-sensitive or "suicide" genes, such as herpes simplex thymidine kinase (TK), p53, and bcl-xs.

[0179] Such therapies can be used to treat and / or prevent diseases and / or conditions. In one implementation, the disease or condition is a hereditary disease or condition. In another implementation, the disease or condition is a sporadic disease or condition. Examples of such diseases or conditions include, but are not limited to, cancers (such as lymphoma, leukemia, or multiple myeloma), lysosomal storage diseases, β-thalassemia, cerebral adrenoleukodystrophy, sickle cell disease, hemophilia, Fanconianemia, 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, and macular degeneration.

[0180] The term "gene therapy" encompasses techniques that modify a subject's genes to treat, cure, or prevent diseases or conditions. Gene therapy can work through several mechanisms: for example, replacing a disease-causing gene with a healthy copy of the gene, inactivating a malfunctioning disease-causing gene, or introducing a new or modified gene into the body to help treat the disease. The transferred genetic material can alter the way cells produce individual proteins or proteomes.

[0181] "Therapies based on retroviridae viruses" can be immunotherapy. "Immunotherapy" encompasses the treatment or prevention of conditions or diseases involving the activation, enhancement, reduction, suppression, or desensitization of the immune system. Therapies based on retroviridae viruses can be vaccines. Preferably, the condition or disease is an autoimmune condition, an allergic reaction, or cancer.

[0182] "Therapies based on retroviral viruses" can be oncolytic virus therapy. "Oncolytic virus therapy" refers to a form of immunotherapy that uses a replicating virus to infect and destroy cancer cells. Preferably, the capable virus specifically attacks tumor cells but not healthy cells.

[0183] The premix may be administered to the subject in any amount and via any route of administration for the effective prevention and / or treatment of disease and / or symptoms.

[0184] The term "subject" includes any organism to which the premix according to the invention may be administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include any living organism, such as animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, and humans). Preferably, the subject requires a therapy based on a retroviridae virus. The term "subject requiring a therapy based on a retroviridae virus" includes individuals who will benefit from a therapy based on a retroviridae virus.

[0185] The term "improved efficacy" for retroviral virus-based therapies includes the following meaning: the therapy achieves the desired clinical outcome with a lower dosing regimen, thereby reducing undesirable side effects, using a retroviral virus-based treatment. Those skilled in the art will be able to select appropriate assays to measure the expected clinical outcome of the agent.

[0186] In one implementation, efficacy is improved due to enhanced transduction resulting from combining retroviridae viruses with albumin to form a premix.

[0187] In one implementation, the premix is ​​administered to the subject immediately after its formation.

[0188] In another implementation, the premix is ​​administered to the subject within a timeframe of approximately 30 seconds to approximately 36 hours after the formation of the premix, such as approximately 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, or 60 seconds to approximately 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 28 hours, 32 hours, 33 hours, 34 hours, 35 hours, or 36 hours.

[0189] In a clinical setting, it may be impractical for healthcare professionals or other staff to expose the premix to cells within seconds or minutes. Therefore, premixing albumin and retroviridae viruses and storing the premix for extended periods (including freezing, e.g., at approximately -15°C to approximately -90°C, or at approximately -15°C to approximately -25°C, or at approximately -70°C to approximately -90°C, or at approximately -80°C or approximately -20°C) can simplify the clinical translation of albumin-enhanced retroviridae virus transduction.

[0190] In yet another implementation, the premix is ​​applied to the subject no more than about 30 seconds after the premix is ​​formed, or no more than about 5 minutes after the premix is ​​formed, such as no more than about 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, or 60 seconds, or no more than about 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5 minutes.

[0191] In one embodiment, the premix is ​​stored at a temperature of about 0°C to about 35°C, such as about 0°C to about 5°C or about 15°C to about 35°C, or at room temperature, such as 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C or 20°C to 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C, and then administered to a subject.

[0192] In one embodiment, the premix is ​​stored at the following temperatures: i) from about 15°C to about 35°C, or at room temperature, for a period of about 30 seconds to about 36 hours; or ii) from about 0°C to about 5°C for a period of about 30 seconds to about 72 hours, and then administered to a subject, for example, 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, and then administered to a subject. When stored at about 15°C to about 35°C (e.g., room temperature), the time period can be from about 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds or 60 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes to about 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 33 hours, 34 hours, 35 hours or 36 hours. When storing from approximately 30 seconds to approximately 72 hours, the time period can be approximately 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds or 60 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes or 50 minutes, 1 hour, 2 hours, 3 hours or 4 hours to approximately 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours or 72 hours.

[0193] Retroviridae viruses are commonly used in therapy, and particularly in cell therapy. Therefore, in a third aspect, the present invention provides a method for generating a cell therapy in vitro, wherein the cell therapy comprises cells transduced by a retroviridae virus, the method comprising: (i) providing cells to be transduced by the retroviridae virus, and (ii) subjecting the cells in vitro to the method of the first aspect of the present invention to generate the cell therapy. The present invention also includes cell therapies thus generated for treating subjects in need, as further described below.

[0194] The term "cell therapy" encompasses techniques that transfer intact, living cells into a subject to treat, cure, or prevent a disease or condition. These cells are typically modified using a viral vector before being administered to the subject. In some implementations, the cells are derived from the subject (autologous cells) or a donor (allogeneic cells).

[0195] Cells used in cell therapy can be classified according to their potential to be converted into different cell types. Cells to be transduced by retroviral viruses can 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, cells can 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 with respect to the first aspect of the invention.

[0196] In one implementation, gene therapy and cell therapy can be combined to treat hereditary diseases. Stem cells are altered via gene therapy in culture (ex vivo). The altered cells are then administered to a subject.

[0197] Chimeric antigen receptors (CARs) engineered with three distinct domains (antigen recognition, co-stimulatory signaling, and T-cell signaling) can be introduced into T cells using lentiviral vectors. Cells expressing the modified receptor recognize antigens of interest and utilize the potent cytotoxic activity of T cells to attack tumor cells. In one embodiment, CAR T-cell therapy targets the CD19 antigen, a protein expressed on B cells and B-cell malignancies.

[0198] A fourth aspect of the invention provides a retroviral-based cell therapy for treating a subject, wherein the cell therapy is derived according to a third aspect of the invention.

[0199] It should also be understood that the present invention includes a method of treating a subject with a cell therapy, wherein the cell therapy comprises cells transduced by a retroviridae virus, the method comprising: (i) providing cells to be transduced by a retroviridae virus, (ii) performing the method of the first aspect of the present invention on the cells in vitro to produce the cell therapy, and (iii) administering the cell therapy to a subject in need.

[0200] Preferred selection of subjects includes those described above with respect to the second aspect of the invention. It should be understood that cell therapy in the context of the third aspect of the invention can be considered a type of retroviral virus-based therapy as described above, wherein the retroviral virus-based therapy comprises cells transduced by a retroviral virus.

[0201] Preferred options for the timing of forming the premix and adding it to the cells include those described above with respect to the first aspect of the invention.

[0202] A fifth aspect of the invention provides a retroviridae-based therapy for treating a subject, wherein the retroviridae virus is combined with albumin to form a premix, which is then administered to the subject.

[0203] It should also be understood that the present invention includes a method for treating a subject with a therapy based on a retroviridae virus, the method comprising: (a) forming the premix; and (b) administering the premix to the subject.

[0204] The term "treatment" or "treating" includes the following meanings: achieving a therapeutic or preventative outcome, which includes, and preferably includes, beneficial or desired clinical results. Such beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of cancer cells or other diseased tissues (or destroying such cancer cells or other diseased tissues), reducing symptoms caused by the disease, improving the quality of life of those suffering from the disease, reducing the dosage of other medications required to treat the disease, delaying the progression of the disease, and / or prolonging the individual's survival.

[0205] Preferred selection of subjects includes those described above with respect to the second aspect of the invention.

[0206] Preferred options for the timing of the formation of the premix and its administration to the subjects include those described above with respect to the first aspect of the invention.

[0207] In one implementation of the second or fifth aspect, the therapy based on retroviral viruses is immunotherapy, such as oncolytic virus therapy, or gene therapy, or a vaccine.

[0208] A sixth aspect of the invention provides the use of albumin for forming a premix to enhance the transduction of retroviridae viruses into cells, wherein albumin is combined with retroviridae viruses to form the premix prior to contact with cells. In one embodiment, cells are contacted in vivo. In another embodiment, cells are contacted ex vivo, such as in vitro.

[0209] A seventh aspect of the invention provides the use of a premix for enhancing the transduction of retroviridae viruses into cells, wherein the premix comprises albumin and a retroviridae virus, and the premix is ​​formed prior to contact with cells. In one embodiment, the cells are contacted in vivo. In another embodiment, the cells are contacted ex vivo, such as in vitro.

[0210] In one embodiment of the sixth or seventh aspect of the invention, the premix is ​​formed immediately before contact with the cells.

[0211] In one embodiment of the sixth or seventh aspect of the invention, the premix is ​​formed from about 30 seconds to about 36 hours prior to contact with cells, such as about 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds or 60 seconds to about 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 28 hours, 32 hours, 33 hours, 34 hours, 35 hours or 36 hours.

[0212] In another embodiment of the sixth or seventh aspect of the invention, the premix is ​​formed no more than about 30 seconds before contact with the cells, or no more than about 5 minutes before contact with the cells, such as no more than about 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds or 60 seconds, no more than about 1 minute, 2 minutes, 3 minutes, 4 minutes or 5 minutes.

[0213] In yet another embodiment of the sixth or seventh aspect of the invention, the premix is ​​stored at temperatures ranging from about 0°C to 35°C, or at room temperature, such as 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C to 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C, for a period of about 30 seconds to about 72 hours; or (ii) from about 15°C to 35°C. (iii) At approximately 0°C to approximately 5°C, such as 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C or 25°C to 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C, for a period of approximately 30 seconds to approximately 36 hours; or (iii) at approximately 0°C to approximately 5°C, such as 0°C, 0.5°C, 1.0°C, 1.5°C, 2.0°C, 2.5°C or 3.0°C to 2.0°C, 2.5°C, 3.0°C, 3.5°C, 4.0°C, 4.5°C or 5°C, for a period of approximately 30 seconds to approximately 72 hours; then contact the cells. The time period from approximately 30 seconds to approximately 72 hours can be approximately 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds or 60 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes or 50 minutes, 1 hour, 2 hours, 3 hours or 4 hours to approximately 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours or 72 hours. The time period from approximately 30 seconds to approximately 36 hours can be approximately 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds or 60 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes to approximately 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 28 hours, 32 hours, 33 hours, 34 hours, 35 hours or 36 hours.

[0214] In one embodiment of the second, third, fourth, fifth, or sixth aspect of the present invention, the premix is ​​as defined in the first aspect of the present invention, and / or the albumin is as defined in the first aspect of the present invention; and / or the retroviridae virus is as defined in the first aspect of the present invention.

[0215] 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.

[0216] This article also discloses a cell-free composition comprising albumin and retroviral viruses.

[0217] This article also discloses a complete kit containing albumin and retroviridae viruses, wherein the albumin and retroviridae viruses are cell-free.

[0218] In one embodiment of the above disclosure, the composition is a premix as defined in the first aspect of the invention; and / or albumin as defined in the first aspect of the invention; and / or a retroviridae virus as defined in the first aspect of the invention.

[0219] This document further discloses the above-disclosed compositions or kits for use in pharmaceuticals. The above-disclosed compositions or kits can be used in pharmaceuticals because the presence of albumin can enhance the transduction of retroviral virus-based therapies.

[0220] This article also discloses the compositions or kits disclosed above for use in gene therapy.

[0221] This article also discloses the compositions or kits disclosed above for use in immunotherapies, such as oncolytic virus therapy.

[0222] This article also discloses the above-disclosed compositions or kits for use as vaccines.

[0223] This document further discloses the methods, uses, compositions, or kits disclosed above, wherein the premix does not contain any of the following: anions having multiple negative charges carried on a flexible backbone, such as any of polyvalent carboxylic acids (e.g., citrate, fumarate, tartaric acid, α-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 inorganic anions.

[0224] The term "anion having a plurality of negative charges carried on a flexible backbone" includes the following meanings: any flexible backbone carrying two or more negative charges, such as three or more, four or more, or five or more negative charges. Therefore, the anion can be multivalent, such as divalent, trivalent, or tetravalent. In one embodiment, the plurality of negative charges carried on the flexible backbone are discontinuous. In another embodiment, the plurality of negative charges carried on the flexible backbone are continuous. The term "flexible backbone" includes the following meanings: a backbone connecting atoms carrying negatively charged groups with single covalent bonds, such that rotational degrees of freedom exist between the negative charges.

[0225] Examples of suitable anions having multiple negative charges carried on a flexible framework include polyvalent organic anions, such as organic carboxylic acids. For example, the anion can be a divalent or trivalent carboxylic acid. In one embodiment, the anion is an intermediate of the citric acid cycle, such as any one of citrate, isocitrate, α-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, or oxaloacetate. In another embodiment, the anion is any one of tartaric acid, maleic acid, succinic acid, aconitate, adenosine triphosphate, or sodium tripolyphosphate. Inorganic anions are also included.

[0226] This document also discloses the methods, uses, compositions or kits disclosed above, wherein the premix does not contain buffers from the following list: citric acid, tartaric acid, lactic acid or potassium citrate.

[0227] This document also discloses the methods, uses, compositions or kits disclosed above, wherein the premix does not contain lipopolysaccharides.

[0228] This document also discloses the methods, uses, compositions or kits disclosed above, wherein the premix does not contain polyethylene glycol (PEG).

[0229] This document also discloses the methods, uses, compositions or kits disclosed above, wherein the premix does not contain 5.0 mM to 20 mM PBS, pH 6.5-7.8, 0.25 mM to 25 mM HEPES, 0.01 mM to 1 mM MgCl2, or 0.01 mM to 1 mM CaCl2.

[0230] This document also discloses the methods, uses, compositions, or kits disclosed above, wherein the premix does not contain 5.0 mM to 20 mM PBS, pH 6.5-7.8, 0.25 mM to 25 mM HEPES, 0.01 mM to 1 mM MgCl2, 0.01 mM to 1 mM CaCl2, and does not contain sucrose, L(+)-glutamic acid or L(+)-glutamic acid monosodium salt or a mixture of L(+)-glutamic acid / L(+)-glutamic acid monosodium salt.

[0231] This document also discloses the methods, uses, compositions or kits disclosed above, wherein the premix does not contain a cationic polymer selected from the group consisting of: polyethyleneimine (PEI), polylysine, polyornithine, polygluconine, cyclodextrin, chitosan, histone, collagen, activated and / or unactivated dendritic polymers.

[0232] This article also discloses the methods, uses, compositions or kits disclosed above, wherein the albumin is not combined with a pre-formed complex of lentivirus and PEI (polyethyleneimine).

[0233] This document further discloses the methods, uses, compositions, or kits disclosed above, wherein albumin is not combined with a pre-formed complex or polypolymer of DNA and a cationic polymer selected from the group consisting of: polyethyleneimine (PEI), polylysine, polyornithine, polygluconine, cyclodextrin, chitosan, histone, collagen, activated and / or unactivated dendritic polymers.

[0234] This document also discloses the methods, uses, compositions, or kits disclosed above, wherein the premix does not contain Lenti-X. ® Culture medium (Lenti-X 293T cell line growth medium, (Takara Bio Inc)), optionally containing Lenti-X ® The culture medium consisted primarily of 90% Dulbecco's Modified Eagle's Medium (DMEM), 4.5 g / L glucose, 4 mM L-glutamine, 3.7 g / L sodium bicarbonate, 10% tetracycline-free fetal bovine serum, and optionally 1 mM sodium pyruvate.

[0235] This document also discloses the methods, uses, compositions or kits disclosed above, wherein the premix does not contain histidine hydrochloride.

[0236] This document also discloses the methods, uses, compositions or kits disclosed above, wherein the premix does not contain one or more of Tris-HCl buffer, B27 serum-free additive and / or CD-lipid concentrate (e.g., all of them).

[0237] This document further discloses the methods, uses, compositions, or kits disclosed above, wherein the premix does not contain any or more of the following transduction enhancers: silibinin, midostaurin, amphotericin B, nystatin, everolimus, deoxyribonucleoside, BAB-type triblock copolymers (such as PEG-PCL-PEG, PEG-PLGA-PEG, and PEG-PLA-PEG), resveratrol, prostaglandin E2, and poloxamer. ® F108, Poloxamer 388, Dimethyl sulfoxide (DMSO), Ruxolitinib (Jakavi), Fludarabine, Lentiboost ® And their combinations.

[0238] This document also discloses the methods, uses, compositions or kits disclosed above, wherein the premix does not contain one or more of HEPES buffered saline, CaPO4, Tris:EDTA and / or CaCl2.

[0239] This article also discloses the methods, uses, compositions or kits disclosed above, wherein the methods or uses are not limited to increasing the stability of retroviridae viruses.

[0240] It should be understood that methods for enhancing the transduction of retroviridae viruses into cells, as described herein, can be used to reduce the toxicity of retroviridae virus-based therapies, for example, by minimizing or avoiding the need for the use of known transduction enhancers that may be toxic. Therefore, the present invention also provides a method for reducing the side effects of retroviridae virus-based therapies, the method comprising combining the retroviridae virus with albumin to form a premix; and administering the premix to a subject. A method for reducing the side effects of retroviridae virus-based therapies is also provided, the method comprising combining the retroviridae virus with albumin to form a premix, ex vivo contact with cells transduced by the retroviridae virus to be used, and administering the transduced cells to a subject in need. Preferred choices for retroviridae viruses, albumin, and premixes include those described above with respect to the first aspect of the invention. Preferred choices for subjects include those described above with respect to the second or third aspect of the invention.

[0241] It should also be understood that methods for enhancing the transduction of retroviral viruses into cells, as described herein, can be used to reduce the dose of retroviral virus-based therapies (e.g., reducing the number of viral particles administered to a subject and / or reducing the volume of the dose), optionally while maintaining or increasing the efficacy of the therapy. Therefore, this method can be used to achieve desired clinical outcomes using lower dosing regimens of retroviral virus-based therapies. Accordingly, the present invention also provides a method for reducing the dose of a retroviral virus-based therapy, comprising combining a retroviral virus with albumin to form a premix, and administering the premix to a subject in need. A method for reducing the dose of a retroviral virus-based therapy is also provided, comprising combining a retroviral virus with albumin to form a premix, ex vivo contact with cells transduced with the retroviral virus to be used, and administering the transduced cells to a subject in need. Preferred choices for retroviral viruses, albumin, and premixes include those described above with respect to the first aspect of the invention. Preferred choices for subjects include those described above with respect to the second or third aspect of the invention.

[0242] Similarly, it should be understood that methods for enhancing the transduction of retroviridae viruses into cells, as described herein, can be used to reduce the overall cost of retroviridae virus-based therapies, for example, by using lower dosing regimens of retroviridae virus-based therapies to achieve desired clinical outcomes. Therefore, the present invention also includes a method for reducing the cost of retroviridae virus-based therapies, comprising combining a retroviridae virus with albumin to form a premix, and administering the premix to a subject in need. A method for reducing the cost of retroviridae virus-based therapies is also provided, comprising combining a retroviridae virus with albumin to form a premix, ex vivo contact with cells transduced with the retroviridae virus to be used, and administering the transduced cells to a subject in need. Preferred choices for retroviridae viruses, albumin, and premixes include those described above with respect to the first aspect of the invention. Preferred choices for subjects include those described above with respect to the second or third aspect of the invention.

[0243] When the term “comprising” is used in the claims and / or description, the word “a or an” may mean “a”, but it is also consistent with the meanings of “one or more”, “at least one” and “one or more”.

[0244] When we refer to a range, such as the temperature range of "0°C to 35°C", it includes the meaning of about or approximately 0°C to about or approximately 35°C.

[0245] The enumeration or discussion of previously disclosed documents in this specification should not be construed as an admission that such documents are part of the prior art or common knowledge.

[0246] Preferred, non-limiting examples embodying certain aspects of the invention will now be described with reference to the accompanying drawings.

[0247] Example Implementation Plan

[0248] 1. A method for enhancing the transduction of retroviral viruses into cells, the method comprising:

[0249] (i) combining the retroviridae virus with albumin to form a premix; and

[0250] (ii) Contact the cells with the premix formed in step (i).

[0251] 2. The method according to implementation scheme 1, wherein step (ii) is performed immediately after step (i).

[0252] 3. The method according to embodiment 1 or 2, wherein step (ii) is performed within a time range of approximately 30 seconds to approximately 36 hours after step (i).

[0253] 4. The method according to any one of the foregoing embodiments, wherein step (ii) is performed no more than about 30 seconds after step (i), or no more than about 5 minutes after step (i).

[0254] 5. The method according to any one of the foregoing embodiments, wherein the premix formed in step (i) is stored at a temperature of about 0°C to about 35°C or at room temperature, and then the cells are contacted in step (ii).

[0255] 6. The method according to any one of the foregoing embodiments, wherein the premix formed in step (i) is stored at a temperature of about 15°C to about 35°C or at room temperature for a period of about 30 seconds to about 36 hours, and then the cells are contacted in step (ii).

[0256] 7. The method according to any one of embodiments 1 to 5, wherein the premix formed in step (i) is stored at a temperature of about 0°C to about 5°C for a period of about 30 seconds to about 72 hours, and then the cells are contacted in step (ii).

[0257] 8. The method according to any one of embodiments 1 to 7, wherein the cell is a dividing cell or a non-dividing cell.

[0258] 9. The method according to any one of embodiments 1 to 8, wherein the cell is a eukaryotic cell.

[0259] 10. The method according to embodiment 9, wherein the cell is an animal cell, a fungal cell, or a plant cell.

[0260] 11. The method according to embodiment 10, wherein the animal cell is a mammalian cell, fish cell, insect cell, reptile cell, amphibian cell, or avian cell.

[0261] 12. The method according to any one of embodiments 8 to 11, wherein the cell is a cell line.

[0262] 13. The method according to embodiment 12, wherein the cell line is a human embryonic kidney cell line, T cells (e.g., human or mouse T cells), immortalized T cells (e.g., Jurkat cell line), peripheral blood mononuclear cells (PBMCs), stem cells (e.g., mesenchymal stem cells (MSCs) or hematopoietic stem cells), neurons, cardiomyocytes, fibroblasts, Chinese hamster ovary (CHO) cells, or HMEC-1 cells.

[0263] 14. The method according to any one of the foregoing embodiments, wherein the albumin is derived from a recombinant source or from serum.

[0264] 15. The method according to any one of the foregoing embodiments, wherein the albumin is wild-type albumin or a variant thereof, or ovalbumin or a variant thereof.

[0265] 16. The method according to any one of the foregoing embodiments, wherein the albumin comprises the amino acid sequence of mammalian albumin, optionally human albumin, bovine albumin, canine albumin or mouse albumin.

[0266] 17. The method according to any one of the foregoing embodiments, wherein the albumin is:

[0267] (i) recombinant human albumin; or

[0268] (ii) Human serum albumin.

[0269] 18. The method according to any one of the foregoing embodiments, wherein the albumin is:

[0270] a) Yeast-derived albumin, optionally wherein said yeast is Pichia pastoris such as Pichia pastoris, yeast such as Saccharomyces cerevisiae, Candida albicans, or Kluyveromyces such as Kluyveromyces lactis or Kluyveromyces marx, Hansenula polymorpha, Schizosoma sacchari, Yersinia lipolytica, Adenyrus adenine, Candida albicans such as Candida utilis, or Zygomyces such as Zygomyces bainei; or

[0271] b) Albumin of plant origin, optionally rice-derived albumin.

[0272] 19. The method according to any one of the foregoing embodiments, wherein the albumin is a variant of 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.

[0273] 20. The method according to any one of the foregoing embodiments, wherein the albumin in the premix has a concentration of about 1 mg / mL to about 400 mg / mL, such as about 2 mg / mL to about 400 mg / mL, or about 2.5 mg / mL to about 400 mg / mL, about 3 mg / mL to about 400 mg / mL, about 3.5 mg / mL to about 400 mg / mL, about 4 mg / mL to about 400 mg / mL, about 4.5 mg / mL to about 400 mg / mL, about 4.75 mg / mL to about 400 mg / mL, about 10 mg / mL to about 250 mg / mL, about 95 mg / mL to about 210 mg / mL, about 10 mg / mL to about 100 mg / mL, 2.5 mg / mL to about 100 mg / mL, or about 7.5 mg / mL to about 50 mg / mL, or about 10 mg / mL to about 25 mg / mL.

[0274] 21. The method according to any one of the foregoing embodiments, wherein after step (ii), the albumin in the composition comprising the premix and the cells has a concentration of about 0.01 mg / mL to about 4 mg / mL, such as about 0.01 mg / mL to about 0.025 mg / mL, or about 0.03 mg / mL to about 0.1 mg / mL, or about 0.035 mg / mL to about 0.1 mg / mL, or about 0.04 mg / mL to about 0.1 mg / mL, or about 0.045 mg / mL to about 0.1 mg / mL, or about 0.005 mg / mL to about 0.075 mg / mL, or about 0.075 mg / mL to about 0.5 mg / mL, or about 0.1 mg / mL to about 0.25 mg / mL.

[0275] 22. The method according to any one of the foregoing embodiments, wherein the retroviridae virus is a retrovirinae virus or a foam retrovirinae virus, or any virus derived therefrom.

[0276] 23. The method according to embodiment 22, wherein the orthovirinae virus is a lentivirus or any virus derived therefrom.

[0277] 24. The method according to embodiment 22, wherein the orthovirinae virus is any one of α retrovirus, β retrovirus, δ retrovirus, ε retrovirus, γ retrovirus, or any virus derived therefrom.

[0278] 25. The method according to embodiment 24, wherein the γ retrovirus is any one of murine leukemia virus, Abelson murine leukemia virus, Fland virus, feline leukemia virus, koala retrovirus (KoRV), heterophilic murine leukemia virus-associated virus, or any virus derived therefrom.

[0279] 26. The method according to embodiment 22, wherein the foam retrovirinae virus is any one of bovine foam virus, equine foam virus, feline foam virus, prosimian foam virus, or simian foam virus, or any virus derived therefrom.

[0280] 27. The method according to embodiment 23, wherein the lentivirus is any one of human immunodeficiency virus (HIV) such as HIV-1 or HIV-2, simian immunodeficiency virus (SIV), mouse lentivirus, equine infectious encephalitis virus, equine infectious anemia virus (EIAV), caprine arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BIV) or feline immunodeficiency virus (FIV), or any virus derived therefrom.

[0281] 28. The method according to any one of the foregoing embodiments, wherein the retroviridae virus is any one of a viral particle, a viral vector such as a retroviridae virus vector, or a vector virus.

[0282] 29. The method according to any one of the foregoing embodiments, wherein the retroviridae virus is a live retroviridae virus, an attenuated retroviridae virus, or a live attenuated retroviridae virus.

[0283] 30. The method according to any one of the foregoing embodiments, wherein the retroviridae virus is in the form of a vaccine.

[0284] 31. The method according to any one of the foregoing embodiments, wherein the retroviridae virus in the premix is ​​present in the range of 0.1-100 multiples of infection (MOI).

[0285] 32. The method according to any one of the foregoing embodiments, wherein the premix further comprises a buffer solution.

[0286] 33. The method according to embodiment 32, wherein the buffer solution has a buffering capacity of about pH 6.0 to about pH 8.0.

[0287] 34. The method according to embodiment 32 or 33, wherein the buffer is any of the following: phosphate-buffered saline (PBS), Hanks buffer, Earl balanced salt solution, Tyrode's solution, MOPS buffer, HEPES buffer, or any isotonic solution for injection such as Plasma-Lyte. ® For example, Plasma-Lyte ® 148 or Ringer lactate.

[0288] 35. The method according to any one of embodiments 32 to 34, wherein the premix comprises a buffer solution of 1 mM to 250 mM.

[0289] 36. The method according to any one of the foregoing embodiments, wherein the premix comprises 9.5 mg / mL to 10.5 mg / mL, preferably about 10 mg / mL, albumin (w / v), 20 mM to 30 mM, preferably 23 mM to 26 mM, sodium, 0 mM to 0.03 mM, preferably 0 mM to 0.1 mM, octanoate, wherein the pH is 6 to 7, preferably about 6.5.

[0290] 37. The method according to any one of the foregoing embodiments, wherein the transduction of the retroviridae virus into cells is enhanced by at least 1.5 times compared to the transduction of the retroviridae virus into cells when the retroviridae virus is not premixed with albumin.

[0291] 38. The method according to any one of the foregoing embodiments, wherein the enhancement of transduction is greater than or equal to the enhancement achieved by using transduction provided below:

[0292] (i)Lentiboost ® ;or

[0293] (ii) Contains poloxamer 338 or poloxamer Synperonic ® The transduction enhancer composition of F108, optionally containing poloxamer 338 or poloxamer Synperonic ® The concentration of F108 is from about 50 µg / ml to about 5000 µg / ml, such as from about 500 µg / ml to about 1000 µg / ml or from about 50 µg / ml to about 500 µg / ml; or

[0294] (iii) A transduction-enhancing composition comprising polyacrylamide; or

[0295] (iv) Combination of (ii) and (iii).

[0296] 39. The method according to any one of the foregoing embodiments, wherein the cells are further contacted with one or more transduction enhancers.

[0297] 40. The method according to any one of the foregoing embodiments, wherein enhanced transduction is manifested as an increase in the expression of the transgenic gene within the transduced cell, optionally wherein the increase in the expression of the transgenic gene is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, or at least 10-fold.

[0298] 41. The method according to any one of the foregoing embodiments, wherein the enhanced transduction is manifested as an increase in the activity of the transgenic protein encoded by the transgene within the transduced cell.

[0299] 42. A method for improving the efficacy of a therapy based on a retroviridae virus, the method comprising combining the retroviridae virus with albumin to form a premix; and administering the premix to a subject.

[0300] 43. The method according to embodiment 42, wherein the efficacy is improved due to enhanced transduction caused by combining the retroviridae virus with albumin to form a premix.

[0301] 44. The method according to embodiment 42 or 43, wherein the premix is ​​applied to the subject immediately after its formation.

[0302] 45. The method according to any one of embodiments 42 to 44, wherein the premix is ​​applied to the subject within a time range of about 30 seconds to about 36 hours after the formation of the premix.

[0303] 46. ​​The method according to any one of embodiments 42 to 45, wherein the premix is ​​applied to the subject no more than about 30 seconds after the formation of the premix, or no more than about 5 minutes after the formation of the premix.

[0304] 47. The method according to any one of embodiments 42 to 46, wherein the premix is ​​stored at a temperature of about 0°C to about 35°C, such as about 0°C to about 5°C or about 15°C to about 35°C, or at room temperature, and then applied to the subject.

[0305] 48. The method according to any one of embodiments 42 to 47, wherein the premix is ​​stored at the following temperature:

[0306] i) A period of time lasting from about 30 seconds to about 36 hours at about 15°C to about 35°C, or at room temperature; or

[0307] ii) A period of time from approximately 0°C to approximately 5°C, lasting from approximately 30 seconds to approximately 72 hours.

[0308] It is then applied to the subject.

[0309] 49. A method for generating cell therapy in vitro, wherein the cell therapy comprises cells transduced by a retroviridae virus, the method comprising:

[0310] (i) Providing the cells to be transduced by the retroviridae virus, and

[0311] (ii) The cells are subjected to the method according to any one of embodiments 1 to 41 in vitro to produce the cell therapy.

[0312] 50. A retroviridae-based cell therapy for use in treating a subject, wherein the retroviridae-based cell therapy is generated by the method according to embodiment 49.

[0313] 51. A retroviridae-based therapy for treating a subject, wherein the retroviridae virus is combined with albumin to form a premix, and the premix is ​​subsequently administered to the subject.

[0314] 52. The therapy based on retroviridae virus used according to embodiment 51, wherein the retroviridae virus is combined with albumin to form a premix immediately before administration to the subject.

[0315] 53. A retroviridae-based therapy used according to embodiment 51 or 52, wherein the retroviridae virus is combined with albumin to form a premix over a time range of approximately 30 seconds to approximately 36 hours prior to administration to the subject.

[0316] 54. A retroviridae-based therapy used according to any one of embodiments 51 to 53, wherein the retroviridae virus is combined with albumin to form a premix no more than about 30 seconds before administration to the subject, or no more than about 5 minutes before administration to the subject.

[0317] 55. A retroviral-based therapy according to any one of embodiments 51 to 54, wherein the premix is ​​stored at a temperature of about 0°C to about 35°C, such as about 0°C to about 5°C or about 15°C to about 35°C or room temperature, and then administered to the subject.

[0318] 56. The method according to any one of embodiments 42 to 49 or the therapy based on a retroviridae virus used according to any one of embodiments 51 to 55, wherein the retroviridae virus-based therapy is an immunotherapy, such as oncolytic virus therapy, or wherein the retroviridae virus-based therapy is a gene therapy, or wherein the retroviridae virus-based therapy is a vaccine.

[0319] 57. Use of albumin to form a premix to enhance transduction of retroviridae viruses into cells, wherein the albumin is combined with the retroviridae virus to form the premix prior to contact with the cells.

[0320] 58. Use of a premix for enhancing transduction of retroviridae viruses into cells, wherein the premix comprises albumin and the retroviridae virus, and the premix is ​​formed prior to contact with the cells.

[0321] 59. The use according to embodiment 57 or 58, wherein the premix is ​​formed immediately before contact with the cells.

[0322] 60. The use according to any one of embodiments 57 to 59, wherein the premix is ​​formed within a time range of about 30 seconds to about 36 hours before contacting the cells.

[0323] 61. The use according to any one of embodiments 57 to 60, wherein the premix is ​​formed no more than about 30 seconds or no more than about 5 minutes before contacting the cells.

[0324] 62. The use according to any one of embodiments 57 to 61, wherein the premix is ​​stored at the following temperature:

[0325] (i) a period of time lasting from about 0°C to about 35°C, or at room temperature, for about 30 seconds to about 72 hours; or

[0326] (ii) A period of time lasting from about 30 seconds to about 36 hours at about 15°C to about 35°C, or at room temperature; or

[0327] (iii) A period of time from about 0°C to about 5°C, lasting from about 30 seconds to about 72 hours;

[0328] Then contact the cells.

[0329] 63. The method according to any one of embodiments 42 to 49 or 56, the retroviral virus-based cell therapy according to embodiment 50, or the retroviral virus-based therapy used according to any one of embodiments 51 to 56, or the use according to any one of embodiments 57 to 62, wherein:

[0330] (i) the premix is ​​as defined in any one of embodiments 32 to 36; and / or

[0331] (ii) the albumin as defined in any one of embodiments 14 to 21; and / or

[0332] (iii) The retroviridae virus is as defined in any one of embodiments 22 to 31.

[0333] 64. The use according to any one of embodiments 57 to 62, wherein:

[0334] (i) the cells are as defined in any one of embodiments 8 to 13; and / or

[0335] (ii) The transduction enhancement is as defined in any one of embodiments 37 to 40.

[0336] 65. A cell-free composition comprising albumin and a retroviral virus.

[0337] 66. A kit comprising albumin and a retroviridae virus, wherein the albumin and the retroviridae virus are cell-free.

[0338] 67. The composition according to embodiment 65 or the kit according to embodiment 66, wherein:

[0339] (i) the composition is a premix as defined in any one of embodiments 32 to 36; and / or

[0340] (ii) the albumin as defined in any one of embodiments 14 to 21; and / or

[0341] The retroviridae virus is as defined in any one of embodiments 22 to 31.

[0342] 68. The composition according to embodiment 65 or 67, or the kit according to embodiment 66 or 67, for use in medicine.

[0343] 69. The composition according to embodiment 65 or 67, or the kit according to embodiment 66 or 67, for use in gene therapy.

[0344] 70. The composition according to embodiment 65 or 67, or the kit according to embodiment 65 or 67, for use in immunotherapies such as oncolytic virus therapy.

[0345] 71. The composition according to embodiment 65 or 67, or the kit according to embodiment 66 or 67, is used as a vaccine.

[0346] 72. The method, use, composition, or kit according to any one of the foregoing embodiments, wherein the premix does not contain: anions having a plurality of negative charges carried on a flexible backbone, such as any of the following polyvalent carboxylic acids: citrate, fumarate, tartaric acid, α-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 coenzyme A; or such as inorganic anions.

[0347] 73. The method, use, composition or kit according to any one of the foregoing embodiments, wherein the premix does not contain a buffer from the following list: citric acid, tartaric acid, lactic acid or potassium citrate.

[0348] 74. The method, use, composition or kit according to any one of the foregoing embodiments, wherein the premix does not contain lipopolysaccharides.

[0349] 75. The method, use, composition or kit according to any one of the foregoing embodiments, wherein the premix does not contain polyethylene glycol (PEG).

[0350] 76. The method, use, composition or kit according to any one of the foregoing embodiments, wherein the premix does not contain 5.0 mM to 20 mM PBS, pH 6.5-7.8, 0.25 mM to 25 mM HEPES, 0.01 mM to 1 mM MgCl2, or 0.01 mM to 1 mM CaCl2.

[0351] 77. The method, use, composition, or kit according to any one of the foregoing embodiments, wherein the premix does not contain 5.0 mM to 20 mM PBS, pH 6.5-7.8, 0.25 mM to 25 mM HEPES, 0.01 mM to 1 mM MgCl2, 0.01 mM to 1 mM CaCl2, and does not contain sucrose, L(+)-glutamic acid or L(+)-glutamic acid monosodium salt or a mixture of L(+)-glutamic acid / L(+)-glutamic acid monosodium salt.

[0352] 78. The method, use, composition or kit according to any one of the foregoing embodiments, wherein the premix does not contain a cationic polymer selected from the group consisting of: polyethyleneimine (PEI), polylysine, polyornithine, polygluconine, cyclodextrin, chitosan, histone, collagen, activated and / or unactivated dendritic polymers.

[0353] 79. The method, use, composition or kit according to any one of the foregoing embodiments, wherein the albumin is not combined with a pre-formed complex of lentivirus and PEI (polyethyleneimine).

[0354] 80. The method, use, composition, or kit according to any one of the foregoing embodiments, wherein albumin is not combined with a pre-formed complex or polypolymer of DNA and a cationic polymer selected from the group consisting of: polyethyleneimine (PEI), polylysine, polyornithine, polygluconine, cyclodextrin, chitosan, histone, collagen, activated and / or unactivated dendritic polymers.

[0355] 81. The method, use, composition, or kit according to any one of the foregoing embodiments, wherein the premix does not contain Lenti-X. ® Culture medium, optionally containing Lenti-X ® The culture medium consisted primarily of 90% Durbeco Modified Eagle Medium (DMEM), 4.5 g / L glucose, 4 mM L-glutamine, 3.7 g / L sodium bicarbonate, 10% tetracycline-free fetal bovine serum, and optionally 1 mM sodium pyruvate.

[0356] 82. The method, use, composition or kit according to any one of the foregoing embodiments, wherein the premix does not contain histidine hydrochloride.

[0357] 83. The method, use, composition or kit according to any one of the foregoing embodiments, wherein the premix does not contain one or more (e.g., all) of Tris-HCl buffer, B27 serum-free additive and / or CD-lipid concentrate.

[0358] 84. The method, use, composition, or kit according to any one of the foregoing embodiments, wherein the premix does not contain any or more of the following transduction enhancers: silymarin, midottolin, amphotericin B, nystatin, everolimus, deoxyribonucleoside, BAB-type triblock copolymers such as PEG-PCL-PEG, PEG-PLGA-PEG, and PEG-PLA-PEG, resveratrol, prostaglandin E2, poloxamer, or synthesized protein. ® F108, Poloxamer 388, Dimethyl sulfoxide (DMSO), Ruxotinib (Jakavi), Fludarabine (Fludara), Lentiboost ® And their combinations.

[0359] 85. The method, use, composition or kit according to any one of the foregoing embodiments, wherein the premix does not contain one or more of HEPES buffered saline, CaPO4, Tris:EDTA and / or CaCl2.

[0360] The present invention is further illustrated by the following embodiments, which should not be construed as limiting the scope of the invention.

[0361] Example 1: Albumin-enhanced lentiviral transduction

[0362] Materials and methods

[0363] Cell lines and lentiviruses

[0364] Human embryonic kidney 293 cells (HEK293) were provided by the European Certified Cell Culture Collection (ECACC) and grown in Eagle's Minimum Essential Medium (EMEM, Gibco, UK) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 2 mM L-glutamine (Gibco, UK), 1% non-essential amino acids (Gibco, UK), and 1% penicillin / streptomycin (Gibco, USA). Cells were maintained at 37°C in a humid environment with 5% CO2. Jurkat cells were provided by ECACC and grown in RPMI 1640 (Gibco, UK) supplemented with 10% FBS, 2 mM L-glutamine (Gibco, UK), and 1% penicillin / streptomycin (Gibco, USA). Cells were maintained at 37°C in a humid environment with 5% CO2. Control lentiviral particles (product number LV10001, viral concentration approximately 1×10⁻⁶) 8 IU / ml, where “IU” is an infectious unit (SEQ ID NO: 6), provided by Charles River (USA) and composed of green fluorescent protein (GFP) genes under the control of the cytomegalovirus (CMV) promoter.

[0365] Control lentiviral particle (product number LV10001): pLenti-SV40-puro-CMV-GFP-Kan_NGS (SEQ ID NO: 6):

[0366]

[0367] Lentiviral transduction

[0368] HEK293 and Jurkat cells were both used at a rate of 1×10 4 Cells / well were seeded in 96-well plates and allowed to incubate overnight. Generally, the MOI used for transduction was 5. Recombinant human serum albumin, Recombumin... ® Elite (Albumedix, UK) was diluted 1 / 10 in PBS (Gibco, UK) to produce a 10 mg / ml solution. Ten μL of diluted albumin or PBS was mixed with the lentivirus to form a premix. The final albumin concentrations in the premix were 8.9 mg / ml for HEK293 and 7.1 mg / ml for Jurkat cells. The premix was then added directly to the cells. The cells were incubated at 37°C and 5% CO2 for 48 hours. Lentiboost was then used as needed. ® The polybrene was supplied by Sirion Biotech (Germany) and diluted 1:100 to 1 mg / ml as the final concentration. Polybrene was supplied by Merck Life Sciences (UK) and used at a final concentration of 8 µg / ml. When polybrene was used in transduction with Jurkat cells, after 4 hours of incubation, the polybrene was removed by centrifuging the cells at 400 × g for 10 minutes and replacing the polybrene-containing medium with complete medium. The cells were then incubated at 37°C and 5% CO2 for 48 hours.

[0369] Flow cytometry analysis

[0370] Following transduction, HEK293 cells were removed from the wells by trypsin digestion (Gibco, USA), washed, and resuspended in BDPharmingen staining buffer (BSA). Jurkat cells were washed and resuspended in staining buffer. For flow cytometry analysis, BD Celesta was used. TM Forward and lateral scattering characteristics and GFP light emission were analyzed in 10,000 events using a FITC filter array (530 / 30) on a cell analyzer (BD, USA). The percentage of transduced cells expressing GFP was determined by comparing them with untransduced controls using FlowJo software (Becton Dickinson, USA).

[0371] Statistical analysis

[0372] Statistical significance among multiple groups was evaluated using one-way ANOVA and post-hoc Tukey's test. Unpaired Student's t-tests were used for analysis between two groups.

[0373] Results and discussion

[0374] Effects of albumin and lentiviral premix on HEK293 and Jurkat cell transduction

[0375] Immediately before transducing HEK293 and Jurkat cells, albumin (Recombumin) was added. ® The effect of premixing lentivirus with albumin was investigated. Lentiviral virus and albumin were added to cells in the following order: albumin followed by lentivirus; lentivirus followed by albumin; lentivirus and albumin added to cells simultaneously; or albumin and lentivirus premixed together before being added to cells. This was done to determine whether premixing lentivirus with albumin was necessary to achieve the observed enhanced transduction levels. Figure 1 For HEK293 and Jurkat cells, the final albumin concentrations in the premix were 8.9 mg / ml and 7.1 mg / ml, respectively. When albumin and lentivirus were added simultaneously rather than as part of the premix, the resulting albumin concentration was the same as if they had been added to the premix. Figure 1 The study showed that albumin and lentivirus need to be mixed together in a premix to achieve any transduction enhancement. When albumin and lentivirus were added directly to the cells without premixing, the transduction levels were the same as those of the lentivirus alone; however, when albumin and lentivirus were combined in a premix, the transduction of both was increased by approximately 3-fold in both Jurkat and HEK293 cells.

[0376] Effect of premix time

[0377] The duration of incubation at room temperature for the albumin and lentivirus premix was determined. Stock solutions of albumin and lentivirus, or PBS and lentivirus, were prepared, incubated at room temperature, and added to cells at gradually increasing time intervals. Transduction levels were measured after 48 hours of incubation. The percentage of Jurkat cells expressing GFP remained constant for up to 24 hours when using the albumin premix. Figure 2 The percentage of cells expressing GFP in the PBS premix decreased over time. While the overall number of HEK293 cells expressing GFP did decrease over time, the fold increase in transduction was higher compared to the PBS sample. These data suggest that albumin and lentiviral premixes can be prepared up to 24 hours in advance and still result in higher levels of transduction.

[0378] Different concentrations of Recombumin ® Elite

[0379] A series of albumin concentrations ranging from 1 mg / ml to 100 mg / ml were prepared by dilution in PBS. These different albumin solutions were then used in transduction experiments as described above. The highest albumin concentration used was 25 mg / ml–100 mg / ml. Figure 3 At a final albumin concentration of 17.8 mg / ml–71.4 mg / ml in the premix, Jurkat cells showed a significantly higher percentage of cells expressing GFP. The number of cells expressing GFP then decreased with increasing albumin concentration until 2.5 mg / ml (i.e., an albumin concentration of 1.8 mg / ml in the premix). Concentrations of 2.5 mg / ml and 1 mg / ml (i.e., albumin concentrations of 1.8 mg / ml and 0.7 mg / ml in the premix, respectively) did not show enhanced transduction, as these concentrations produced the same number of transduced cells as lentivirus premixed with PBS. For HEK293 cells, a similar trend in transduction levels was observed, with higher albumin concentrations of 25 mg / ml–100 mg / ml (i.e., albumin concentrations of 22.3 mg / ml–89.3 mg / ml in the premix) producing a higher percentage of cells expressing GFP.

[0380] When testing different concentrations of Recombumin ® In Elite, "x mg / ml" refers to the concentration of the stock albumin used to prepare the premix. After mixing with lentivirus, this yields a premix with albumin concentrations according to Table 1.

[0381] Table 1

[0382] A) Jurkat cells

[0383]

[0384] B) HEK293 cells

[0385]

[0386] Effects of human serum albumin from different sources on transduction

[0387] Albumin from different sources was tested to observe whether it had an effect on transduction levels. Albumin from various sources, such as recombinant human albumin derived from Saccharomyces cerevisiae, was used. ®Elite (“Sacch.”), human serum albumin (“serum-derived”), rice-derived recombinant albumin (“rice”), and Pichia pastoris-derived albumin (“Pichia pastoris”). All transductions were performed using premixes prepared from 25 mg / mL albumin stock solution, resulting in albumin concentrations of 22.3 mg / mL for HEK293 and 17.8 mg / mL for Jurkat cells. Both cell lines tested showed that the albumin source did not affect transduction enhancement. Figure 4 For Jurkat cells, the transduction fold increase was approximately 7.5-fold for all samples, and for HEK293, the transduction fold increase was approximately 5-fold for all samples. This indicates that the source of albumin does not affect the transduction enhancement effect.

[0388] Comparison with Albumin Alternatives

[0389] To determine whether the enhanced transduction effect was limited to human sequence albumin, albumins with sequences from different animals were tested. Recombinant canine albumin (SEQ ID NO: 3) and recombinant mouse albumin (SEQ ID NO: 2) were provided as 100 mg / ml solutions and diluted with PBS to 25 mg / ml. Egg white albumin or “ovalbumin” (SEQ ID NO: 5) and bovine serum albumin (SEQ ID NO: 4) were provided as lyophilized powders and stock solutions were prepared in PBS to 25 mg / ml. Premixing and transduction were performed as described above. For HEK293 and Jurkat cells, the albumin concentrations in the premixes were 22.3 mg / mL and 17.8 mg / mL, respectively. For HEK293 cells ( Figure 5 B), Recombinant human albumin ® Elite (“Sacch.”) and egg white albumin showed similar increases in transduction: 9.3-fold and 9-fold, respectively. Both albumins showed greater increases in transduction than bovine serum albumin, recombinant canine albumin, and recombinant mouse albumin, which showed increases of 7.8-fold, 7.9-fold, and 7.3-fold, respectively. For Jurkat cells, all albumins showed at least a 5-fold increase in transduction compared to PBS. Figure 5 A).

[0390] The impact of MOI

[0391] Transduction was performed using a series of MOIs (1-10) to determine the binding of lentivirus with albumin (Recombumin). ®The question was whether the Elite premix could still achieve transduction enhancement at a range of lentiviral concentrations. For HEK293 and Jurkat cells, the albumin concentrations in the premix were 8.9 mg / mL and 7.1 mg / mL, respectively. For Jurkat cells, the percentage of cells expressing GFP increased with increasing MOI in both the lentiviral sample (PBS) and the albumin-treated sample. However, the fold increase in transduction between the untreated and albumin-treated samples remained relatively consistent, with MOIs of 4.5-fold, 6.3-fold, 6.2-fold, and 4.2-fold for 1, 2, 5, and 10, respectively. Figure 6 A). For HEK293 cells, the fold increase in transduction between untreated and albumin-treated samples also increased with increasing MOI, with MOIs of 1, 2, 5, and 10 being 1.2-fold, 3.0-fold, 1.8-fold, and 2.4-fold, respectively. Figure 6 B).

[0392] The effects of freezing and storing premixes

[0393] To determine the storage conditions of lentiviral albumin (Recombumin) at -80°C ® To determine whether the (Elite) premix had any harmful effects, premixes of lentivirus and albumin were prepared for both HEK293 and Jurkat cells. The albumin concentrations in the premixes were 8.9 mg / mL for HEK293 cells and 7.1 mg / mL for Jurkat cells. The premix was then divided into four equal aliquots. Three aliquots were placed in a -80°C freezer, and the remaining aliquot was used for transduction at time 0. One of the remaining aliquots was removed from the freezer monthly and used for transduction, a process repeated for three months. The percentage of cells expressing GFP was determined. Figure 8 For HEK293 cells at all test time points, the albumin premix produced significantly more GFP-expressing cells than the PBS sample. Between time 0 and 1 month, the percentage of GFP-expressing cells decreased, but this was expected because the time 0 sample did not undergo freeze-thaw cycles, while the 1-, 2-, and 3-month samples all underwent a single freeze-thaw cycle. For the 1-, 2-, and 3-month samples, the percentage of GFP-expressing cells remained constant for both the PBS and albumin premix samples, with the albumin premix sample showing significantly more GFP-expressing cells at all time points. These data suggest that the transduction enhancement benefits of the albumin premix can be maintained for up to 6 months at -80°C storage. The same trend was observed in Jurkat cells (data not shown).

[0394] Comparison with alternative transduction enhancers and lentivirus + albumin combination

[0395] Recombinin will be used ® Transduction with the Elite (Sacch) premix was compared to transduction using commercially available transduction enhancers. Commercially available transduction enhancers were used according to the manufacturer's instructions. For HEK293 and Jurkat cells, the albumin concentrations in the premix were 22.3 mg / mL and 17.8 mg / mL, respectively. When the commercial transduction enhancer was used in combination with the albumin premix, the commercial transduction enhancer was added to the cells, followed by the albumin premix. A synergistic effect was observed in the percentage of GFP-expressing cells obtained when the albumin premix and the commercial transduction enhancer were combined (Figure 7). For HEK293 cells and polybrene (Figure 7B), the albumin premix produced only 10.6% of GFP-expressing cells, and polybrene produced only 3.8% of GFP-expressing cells, compared to 27.4% for the combination of both polybrene and the albumin premix. This is significantly higher than the sum of the individual combinations of polybrene and the albumin premix, indicating a synergistic effect. For HEK293, in addition to the albumin premix, Lentiboost was added. ® No synergistic effect was observed (Figure 7D). The data indicate that Lentiboost ® It had no effect on increasing lentiviral transduction, as lentiviral transduction alone produced 3.29% of cells expressing GFP, and the addition of Lentiboost to the transduction... ® This resulted in 3.06% of cells expressing GFP. This result explains why combining the two transduction enhancers had no effect on the observed GFP levels in cells, with only the albumin premix showing 13.4%, compared to the albumin premix and Lentiboost. ® The combined effect was 12.6%. This result was not significantly different from that of the albumin premix alone, indicating that Lentiboost did not enhance transduction in HEK293 cells.

[0396] For Jurkat cells, targeting Lentiboost ® A synergistic effect was observed with both lentiboost and polyacrylamide. ® Individual transduction enhancers for albumin premix and Lentiboost ® The results yielded 4.13% and 6.01% of cells expressing GFP, respectively, compared to 23% when combined (Fig. 7C). For polybrene, the individual transduction enhancers were effective against albumin premix and Lentiboost. ®The two methods yielded 5.55% and 1.17% of cells expressing GFP, respectively, compared to 7.77% when combined (Figure 7A). These results suggest that combining the two transduction enhancers provides additional advantages when transducing cells.

[0397] The difference between the effects of albumin on lentiviral transduction and on lentiviral stability

[0398] To evaluate albumin (Recombumin) ® Two different time-course experiments were conducted to investigate whether Elite (the lentivirus) enhanced transduction or improved lentiviral stability. In one experiment, an albumin premix was prepared and incubated at room temperature, with samples taken every 2 hours and added to cells for transduction; in the other experiment, lentivirus was stored in PBS at room temperature and mixed with albumin to form a premix, which was then immediately added to cells for transduction. The albumin concentrations in the premixes were 8.9 mg / ml and 7.1 mg / ml for HEK293 and Jurkat cells, respectively.

[0399] Lentivirals are unstable at room temperature, and their ability to transduce cells decreases over time. Lentivirals stored in PBS at room temperature will lose their ability to transduce cells over time, while lentiviruses stored in albumin will remain more stable. For both HEK293 and Jurkat cells, lentiviruses stored in PBS showed decreased transduction efficiency over time compared to samples stored in albumin. Figure 9 Compared to those containing PBS, the transduction efficiency of the premix containing albumin increased fold over time, from 2.1 to 5.53 times for Jurkat cells (8 hours) and from 2.2 to 6.26 times for HEK293 cells (8 hours), indicating that albumin stabilized the lentivirus. However, the observed effects were different when the lentivirus was stored in PBS at room temperature, then combined with albumin to form a premix, and then immediately added to the cells. Figure 10 In this experiment, for both albumin and PBS premix, the percentage of cells expressing GFP decreased over time, although the albumin premix produced a higher percentage of cells expressing GFP than the PBS premix. This indicates that when lentivirus is immediately mixed with albumin and used for transduction, albumin enhances transduction rather than increases stability. For both Jurkat and HEK293 cells, after 4 hours at room temperature, the fold increase in transduction with the albumin premix remained stable compared to the PBS premix. This suggests that albumin is not stabilizing the virus, but rather enhancing transduction. When the virus becomes unstable in PBS at room temperature, albumin is able to rescue transduction by enhancing transduction rather than stabilizing the virus. This contrasts with the sample stored in the albumin premix, which showed an increase in fold increase in transduction over time. Figure 9 Without being bound by any theory, this demonstrates that when combined with lentivirus immediately before transduction into cells, albumin does not stabilize the virus, but rather enhances transduction. These two time-course experiments show that the effect of albumin on lentivirus is 2-fold, depending on how the albumin is utilized. When the lentivirus is stored in albumin before being added to the cells, albumin both stabilizes and enhances transduction; however, if albumin is immediately mixed with the lentivirus and transferred to the cells, it only enhances transduction. This work demonstrates the ability of albumin to be used in novel methods to enhance transduction in cells such as mammalian cells.

[0400] Example 2: Effect of albumin concentration on lentivirus transduction .

[0401] Materials and methods

[0402] Cell lines and lentiviruses

[0403] Human embryonic kidney 293 cells (HEK293) were provided by the European Certified Cell Culture Collection (ECACC) and grown in Eagle's Minimum Essential Medium (EMEM, Gibco, UK) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 2 mM L-glutamine (Gibco, UK), 1% non-essential amino acids (Gibco, UK), and 1% penicillin / streptomycin (Gibco, USA). Cells were maintained at 37°C in a humid environment with 5% CO2. Jurkat cells were provided by ECACC and grown in RPMI 1640 (Gibco, UK) supplemented with 10% FBS, 2 mM L-glutamine (Gibco, UK), and 1% penicillin / streptomycin (Gibco, USA). Cells were maintained at 37°C in a humid environment with 5% CO2. Control lentiviral particles (product number LV10001, viral concentration approximately 1×10⁻⁶) 8 IU / ml, SEQ ID NO: 6) was provided by Charles River (USA) and is composed of green fluorescent protein (GFP) genes under the control of the cytomegalovirus (CMV) promoter.

[0404] Lentiviral transduction

[0405] HEK293 and Jurkat cells were both used at a rate of 1×10 4 Cells / well were seeded in 96-well plates and allowed to incubate overnight. The MOI used for transduction was 5. Recombinant human serum albumin, Recombumin... ®Elite (Albumedix, UK) was diluted in PBS (Gibco, UK) to produce a series of concentrations from 2 mg / ml to 25 mg / ml. Ten μL of diluted albumin or PBS was mixed with the lentivirus to form a premix. The albumin concentration in the premix was 1.4 mg / ml–17.8 mg / mL for Jurkat cells and 1.8 mg / ml–22.3 mg / mL for HEK293 cells. The premix was then added directly to the cells. The cells were incubated at 37°C and 5% CO2 for 48 hours.

[0406] Flow cytometry analysis

[0407] Following transduction, HEK293 cells were removed from the wells by trypsin digestion (Gibco, USA), washed, and resuspended in BDPharmingen staining buffer (BSA). Jurkat cells were washed and resuspended in staining buffer. For flow cytometry analysis, BD Celesta was used. ™ Forward and lateral scattering characteristics and GFP light emission were analyzed in 10,000 events using a FITC filter array (530 / 30) on a cell analyzer (BD, USA). The percentage of transduced cells expressing GFP was determined by comparing them with untransduced controls using FlowJo software (Becton Dickinson, USA).

[0408] Statistical analysis

[0409] Statistical significance among multiple groups was evaluated using one-way ANOVA and post-hoc Tukey's test. Unpaired Student's t-tests were used for analysis between two groups.

[0410] Results and discussion

[0411] The effect of albumin concentration on lentiviral transduction

[0412] A series of albumin concentrations ranging from 2 mg / ml to 25 mg / ml were prepared by dilution in PBS. These different albumin solutions were then used in transduction experiments as described above. This was to determine at what albumin concentration it began to have a positive effect on the observed transduction levels. For Jurkat cells, a significant increase in transduction was observed between lentiviral samples alone and samples containing only 4 mg / ml albumin. Figure 11A). This indicates that albumin does not enhance transduction at albumin concentrations below 4 mg / ml (i.e., albumin concentration in the premix is ​​2.9 mg / ml). For HEK293 cells, there was a significant increase in transduction between lentiviral samples alone and albumin samples at 4.5 mg / ml (i.e., albumin concentration in the premix is ​​4.0 mg / ml). Figure 11 (B) This demonstrates that albumin does not enhance transduction at concentrations below this level. This work is consistent with that of Palesch et al., (2016). Palesch used native HSA as a control in HIV transduction of the TZM-B1 cell line and showed no increase in transduction at concentrations of 0 µg / ml–100 µg / ml. In this example, we have shown that a stock albumin concentration of at least 4 mg / ml–4.5 mg / ml (corresponding to 2.9 mg / ml–4.0 mg / ml albumin concentrations in the premix, and approximately 290 µg / ml–400 µg / ml when in wells) is required to achieve transduction enhancement by albumin.

[0413] When testing the effect of albumin concentration on lentiviral transduction, "x mg / ml" refers to the concentration of the stock albumin used to prepare the premix. After mixing with lentivirus, this yields a premix with albumin concentrations according to Table 2.

[0414] Table 2

[0415] A) Jurkat cells

[0416]

[0417] B) HEK293 cells

[0418]

[0419] Example 3: Albumin-enhanced transduction of lentiviruses into T cells and bone marrow-derived mesenchymal stem cells (BM-MCS)

[0420] Materials and methods

[0421] Cell lines and lentiviruses

[0422] Bone marrow-derived mesenchymal stem cells (BM-MSCs) were provided by RoosterBio (USA) and grown in Dürbeco Modified Eagle Medium (DMEM, Gibco, UK) supplemented with 10% MSC-qualified FBS (Gibco, UK). Cells were maintained at 37°C in a humid atmosphere with 5% CO2. T cells were extracted from donor blood using the StemCell Technologies EasySep Direct Human Tcell Isolation Kit (StemCell Technologies, Canada). After extraction, cells were resuspended in Immunocult-XF T cell expansion medium (StemCell Technologies, Canada) at a concentration of 1 × 10⁻⁶ cells / mL. 5 Cells were seeded at 100 µl / well in 96-well plates. Cells were then activated with Immunocult human CD3 / CD28 T cell activation spheres (StemCell Technologies, Canada) and 300 units of IL-2 (StemCell Technologies, Canada). Control lentiviral particles (product number LV10001, viral concentration approximately 1 × 10⁻⁶) were used. 8 IU / ml, SEQ ID NO: 6) was provided by Charles River (USA) and is composed of green fluorescent protein (GFP) genes under the control of the cytomegalovirus (CMV) promoter.

[0423] Lentiviral transduction

[0424] Bone marrow-derived MSCs were used at a rate of 2 × 10 3 cells / cm 2 Inoculate into 96-well plates and allow to incubate for 48 hours. Generally, the MOI used for transduction is 5. Recombinant human serum albumin, Recombumin... ® Elite (Albumedix, UK) was diluted in PBS (Gibco, UK) to produce a 25 mg / ml solution. Ten μL of diluted albumin or PBS was mixed with the lentivirus to form a premix. The albumin concentration in the premix was 17.8 mg / ml. The premix was then added directly to the cells. The cells were incubated at 37°C and 5% CO2 for 72 hours. T cells were activated and seeded as described above. 48 hours after activation, the cells were transduced as described above. The cells were incubated at 37°C and 5% CO2 for 48 hours. Lentiboost was administered as needed. ®Supplied by Sirion Biotech (Germany) and diluted 1:100 to 1 mg / ml as the final concentration.

[0425] Flow cytometry analysis

[0426] Following transduction, BM-MSC cells were removed from the wells by trypsin digestion (Gibco, USA), washed, and resuspended in BDPharmingen staining buffer (BSA). T cells were washed and resuspended in staining buffer. For flow cytometry analysis, BD Celesta was used. ™ Forward and lateral scattering characteristics and GFP light emission were analyzed in 5,000 events using a FITC filter array (530 / 30) on a cell analyzer (BD, USA). The percentage of transduced cells expressing GFP was determined by comparing them to untransduced controls using FlowJo software (Becton Dickinson, USA).

[0427] Statistical analysis

[0428] The analysis between the two samples was performed using an unpaired Student t-test.

[0429] Results and discussion

[0430] Effects of albumin-lentiviral premix on BM-MSCs and T cell transduction

[0431] Immediately before transducing BM-MSCs and T cells, add albumin (Recombumin) ® The effects of Elite and lentivirus premixing were investigated. Figure 12 Lentiviral virus and albumin were premixed before being added to cells. The albumin concentration in the premix was 17.8 mg / ml. For BM-MSCs, transduction with the premixed sample increased by approximately 1.5-fold compared to the lentivirus-only control. For T cells, transduction increased by approximately 6-fold compared to the lentivirus-only control. This demonstrates that the premixed albumin method has a positive effect on the observed transduction levels of both BM-MSCs and T cells.

[0432] Comparison with alternative transduction enhancer Lentiboost

[0433] Recombinin will be used ® The transduction of the Elite (Sacch) premix and the use of the commercially available transduction enhancer Lentiboost ®The transductions performed were compared, the latter being used according to the manufacturer's instructions. The albumin concentration in the premix was 17.8 mg / ml. When Lentiboost ® When used in conjunction with albumin premix, Lentiboost ® It was added to the cells, followed by the addition of an albumin premix. When the albumin premix was combined with a commercial transduction enhancer, it had a positive effect on the percentage of cells expressing GFP obtained. Figure 12 For BM-MSC cells ( Figure 12 B) and Lentiboost ® Lentiviral cells produced only 3.72% of cells expressing GFP, compared to albumin and Lentiboost. ® Cells expressing GFP were produced at 5.57% and 5.88% respectively. When albumin premix and Lentiboost... ® When combined, the percentage of cells expressing GFP increased to 9.88%, almost the same as Lentiboost alone. ® Twice that of the transduction enhancer. This indicates a strong positive effect on transduction levels when two transduction enhancers are combined in BM-MSC cells.

[0434] For T cells, when albumin premix and Lentiboost ® When combined, a synergistic effect on transduction was observed. In the lentivirus-only case, 1.76% of cells were transduced. This is compared to 10.72% for T cells transduced with albumin premix. For Lentiboost ® When using Lentiboost alone ® No increase in transduction was observed at that time, as only 2.03% of cells expressed GFP. However, when albumin premix and Lentiboost were added... ® When used in combination for transduction, 22.6% of cells expressed GFP. Figure 12 A). The transduction level increased 12.8-fold compared to lentivirus alone. This indicates that, in the case of T cells, the combination of two transduction enhancers has a synergistic effect on the achieved transduction level compared to individual transduction enhancers.

[0435] Example 4: Albumin-enhanced transduction of lentiviruses into hematopoietic stem cells

[0436] Materials and methods

[0437] Cell lines and lentiviruses

[0438] Bone marrow-derived CD34+ cells (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 in a humid atmosphere with 5% CO2. Control lentiviral particles were prepared as described in Examples 1-3.

[0439] Lentiviral transduction

[0440] CD34+ cells were seeded at 5000 cells / well (500 µl volume) in 12-well plates and incubated for 24 hours. Generally, the MOI used for transduction is 5. Recombinant human serum albumin and recombumin were added. ® Elite (Albumedix, UK) was diluted in PBS (Gibco, UK) to produce a 25 mg / mL solution. Ten μL of diluted albumin or PBS was mixed with the lentivirus to form a premix. The albumin concentration in the premix was 18.8 mg / mL. This premix was then added directly to the cells. Twenty-four hours after transduction, an additional 500 µL of complete CD34+ medium was added to the wells, and the cells were incubated for another 48 hours.

[0441] Flow cytometry analysis

[0442] After transduction, cells were detached from the plate using a cell scraper, washed in BD CellWash (BD, USA), and resuspended in BSA staining buffer (BD, USA). For flow cytometry analysis, BD CellWash was used. ™ Forward and lateral scattering characteristics and GFP light emission were analyzed in 10,000 events using a FITC filter array (530 / 30) on a cell analyzer (BD, USA). The percentage of transduced cells expressing GFP was determined by comparing them with untransduced controls using FlowJo software (Becton Dickinson, USA).

[0443] Statistical analysis

[0444] The analysis between the two samples was performed using an unpaired Student t-test.

[0445] Effects of albumin and lentivirus premix on CD34+ transduction

[0446] Recombinin will be used ®CD34+ transduction with the "Elite" premix was compared with transduction in the absence of the premix. Figure 13 ).

[0447] Compared with cells transduced using lentivirus alone, the use of the Recombumin Elite premix resulted in an approximately 1.5-fold increase, with the albumin premix yielding 23.4% of GFP-positive cells, compared to 15.9% with lentivirus alone. This represents a statistically significant (p<0.01) improvement in lentivirus transduction of CD34+ cells.

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Claims

1. A method for enhancing the transduction of retroviral viruses into cells, the method comprising: (i) Combining the retroviridae virus with albumin to form a premix; as well as (ii) Contact the cells with the premix formed in step (i).

2. The method of claim 1, wherein step (ii) is performed as follows: (a) immediately following step (i); or (b) Within a timeframe of approximately 30 seconds to approximately 36 hours after step (i); or (c) No more than about 30 seconds after step (i), or no more than about 5 minutes after step (i).

3. The method according to any one of claims 1 or 2, wherein the premix formed in step (i) is stored at the following temperature: (a) at a temperature of about 0°C to about 35°C, or at room temperature, for a period of about 30 seconds to about 72 hours, followed by contact with the cells in step (ii); and / or (b) at a temperature of about 15°C to about 35°C, or at room temperature, for a period of about 30 seconds to about 36 hours, and then contact the cells in step (ii); or (c) At approximately 0°C to approximately 5°C for a period of approximately 30 seconds to approximately 72 hours, the cells are then contacted in step (ii).

4. The method according to any one of the preceding claims, wherein the cell is: (a) dividing or non-dividing cells; and / or (b) Eukaryotic cells, optionally said cells are animal cells, fungal cells, or plant cells, further optionally said animal cells are mammalian cells, fish cells, insect cells, reptile cells, amphibian cells, or avian cells; and / or (c) Cell lines, optionally said cell lines being human embryonic kidney cell lines, T cells (e.g., human or mouse T cells), immortalized T cells (e.g., Jurkat cell lines), peripheral blood mononuclear cells (PBMCs), stem cells (e.g., mesenchymal stem cells (MSCs) or hematopoietic stem cells), neurons, cardiomyocytes, fibroblasts, Chinese hamster ovary (CHO) cells, or HMEC-1 cells.

5. The method according to any one of the preceding claims, wherein the albumin: (a) derived from a recombinant source or from serum; and / or (b) is wild-type albumin or a variant thereof, or ovalbumin; and / or (c) Contains the amino acid sequence of mammalian albumin, optionally human albumin, bovine albumin, canine albumin, or mouse albumin; and / or (d) is: (i) recombinant human albumin; or (ii) Human serum albumin; and / or (e) is: i) Yeast-derived albumin, optionally wherein said yeast is Pichia pastoris such as Pichia pastoris, yeast such as Saccharomyces cerevisiae, Candida albicans, or Kluyveromyces such as Kluyveromyces lactis or Kluyveromyces marx, Hansenula polymorpha, Schizosaccharomyces cerevisiae, Yersinia lipolytica, Adenyrus adenine, Candida albicans such as Candida utilis, or Zygomyces such as Zygomyces bainii; or ii) Albumin of plant origin, optionally rice-derived albumin; and / or (f) is a variant of 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 the wild-type ovalbumin, respectively.

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

7. The method according to any one of the preceding claims, wherein after step (ii), the albumin in the composition comprising the premix and the cells has a concentration of about 0.01 mg / mL to about 4 mg / mL, such as about 0.01 mg / mL to about 0.025 mg / mL, or about 0.03 mg / mL to about 0.1 mg / mL, or about 0.035 mg / mL to about 0.1 mg / mL, or about 0.04 mg / mL to about 0.1 mg / mL, or about 0.045 mg / mL to about 0.1 mg / mL, or about 0.005 mg / mL to about 0.075 mg / mL, or about 0.075 mg / mL to about 0.5 mg / mL, or 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 virus is a virus of the subfamily Orthorevirus or the subfamily Foaming Retrovirus, or any virus derived therefrom, optionally wherein the Orthorevirus is a lentivirus or any virus derived therefrom, and further optionally wherein the lentivirus is any one of human immunodeficiency virus (HIV) such as HIV-1 or HIV-2, simian immunodeficiency virus (SIV), mouse lentivirus, equine infectious encephalitis virus, equine infectious anemia virus (EIAV), caprine arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BIV), or feline immunodeficiency virus (FIV), or any virus derived therefrom.

9. The method according to any one of the preceding claims, wherein the retroviridae virus in the premix is ​​present in the range of 0.1-100 multiples of infection (MOI).

10. The method according to any one of the preceding claims, wherein the transduction of the retroviridae virus into cells is enhanced by at least 1.5 times compared to the transduction of the retroviridae virus into cells when the retroviridae virus is not premixed with albumin.

11. A method for improving the efficacy of a therapy based on a retroviridae virus, the method comprising combining the retroviridae virus with albumin to form a premix; and administering the premix to a subject.

12. A method for generating cell therapy in vitro, wherein the cell therapy comprises cells transduced by a retroviridae virus, the method comprising: (i) Providing the cells to be transduced by the retroviridae virus, and (ii) The cells are subjected to the method according to any one of claims 1 to 10 in vitro to produce the cell therapy.

13. A retroviridae-based cell therapy for use in treating subjects, wherein the retroviridae-based cell therapy is produced by the method of claim 12.

14. A retroviridae-based therapy for treating a subject, wherein the retroviridae virus is combined with albumin to form a premix, and the premix is ​​subsequently administered to the subject.

15. Use of albumin to form a premix to enhance transduction of retroviridae viruses into cells, wherein the albumin is combined with the retroviridae virus to form the premix prior to contact with the cells.

16. Use of a premix for enhancing transduction of retroviridae viruses into cells, wherein the premix comprises albumin and the retroviridae virus, and the premix is ​​formed prior to contact with the cells.

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