LU103239B1 - Baculoviral vector system for delivery of heterologous gene products into mammalian cells - Google Patents

Abstract

The invention relates to a baculoviral vector system for improved delivery of DNA, RNA and protein into mammalian cells, including primary cells, tissues, and whole mammalian organisms.

Description

Our Ref.: G 0180 LU Date: 6 February 2024 LU103239

Applicant: GBiotech Särl

Baculoviral vector system for delivery of heterologous gene products into mammalian cells

Field of the Invention

The invention relates to a baculoviral vector system for improved delivery of DNA, RNA and protein into mammalian cells, including primary cells, tissues, and whole mammalian organisms.

Background of the Invention

Recombinant Protein Expression, Nucleic Acid and Protein Delivery Systems: A vast number of expression systems are used to produce recombinant proteins. Cell based expression systems include those utilizing bacteria, yeast, insect cells or mammalian cells as hosts. A commonly used system is the baculovirus expression vector system. Baculoviruses are enveloped viruses that contain a double stranded circular DNA genome of 80-180 kbp, encoding 90-180 putative proteins (Rohrman, 2019). Baculoviruses have highly species- specific tropisms among invertebrate cells and are not known to replicate in mammalian or other vertebrate animal cells, nor do they cause known pathologies in mammals. For reasons of their safety and exceptional production yield in cultured insect cells, natural baculoviruses have been modified to function as a heterologous production platform for biotechnology applications. Baculoviruses infect a range of insect cell culture lines used throughout the biotechnology research community, including SF9 cells, SF-21 cells, and High Five (BTI-Tn- 5B1-4) cells. Due to its safety, speed, and straightforward molecular biology required to create heterologous expression vectors, the baculovirus expression system has become one of the most widely used systems for production of recombinant proteins. Another key advantage of the baculovirus system is its capacity to accommodate large (= 38 kbp) DNA inserts (Van Oers et al., 2015), which enables the system to be used for delivery of multiple expression cassettes into insect and mammalian cells e.g. the MultiBac™ and

MultiBacMam™ respectively, systems (Geneva Biotech, Pregny-Chambésy, Switzerland).

Alternative viral vector systems such as AAV and lentivirus have a strictly limited cargo capacity only enabling them to carry single expression cassettes due to physical constraints imposed by their respective viral particle geometry. Baculovirus expression systems are used to produce heterologous proteins for fundamental research, diagnostics, vaccines, and for drug discovery. Vaccines manufactured with baculovirus include the registered products

FluBlok®, Cervarix® and Provenge®, and several additional baculovirus-produced protein 1 therapeutics are in clinical trials. Baculovirus is also used to produce recombinant adeno- LU103239 associated virus (rAAV) vectors for gene therapy. Baculovirus-produced Glybera® was the first rAAV gene therapy approved for use in humans, and numerous baculovirus-produced rAAV candidates are in clinical trials.

Baculovirus infection of host cells can be divided to three distinct phases: “early” (0—6h post- infection (p.i.)), “late” (6—24h p.i.) and “very late” (18-24 to 72h p.i.). The baculovirus “very late” promoters display extremely high rates of transcription relative to host insect cell promoters. Therefore, the basic idea behind prior art baculovirus expression of recombinant proteins in insect cells is that the DNA sequence coding for a recombinant protein of interest is shuttled into the baculovirus genome under the control of such a “very late” promoter, which results in high levels of expression of the recombinant protein during (very late stages of) infection of the cultured (host) insect cell. The baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) is by far the most commonly utilized virus in this system.

Several technological improvements have eliminated the original tedious procedures required to create and culture recombinant baculoviruses. Modern baculovirus expression systems allow for recombinant genes to be shuttled into the baculovirus genome through recombination of baculovirus DNA and recombinant gene-containing plasmids in insect cells, in bacteria, or in vitro. Using any of these methods, recombinant protein-encoding viruses are generated to infect insect cell cultures. There are numerous commercial systems available for expressing recombinant proteins using baculovirus, including MultiBac™ (Geneva

Biotech), flashBAC™ (Oxford Expression Technologies), BacPAK™ (BD Biosciences

Clontech), BacVector® 1000/2000/3000 (Novagen®), Bac-to-Bac® (Invitrogen™), and

BaculoDirect™ (Invitrogen™). All of these systems are based on the principle of expressing recombinant proteins by placing them under the control of the very late baculovirus promoters polh or p10. None of these systems allow for co-delivery of heterologous DNA, heterologous RNA and heterologous protein into mammalian cells. “Baculovirus Display” (Mäkelä et al. 2008) is a method whereby heterologous peptides and/or proteins are expressed as fusion proteins with native or heterologous baculoviral envelope proteins, typically utilizing the major envelope glycoprotein gp64, or foreign membrane-derived counterparts. It allows incorporation of heterologous proteins onto the surface of baculovirus particles and thereby delivery into infected or transduced cells. The method is typically used for antigen presentation and creation of complex libraries. Baculovirus Display does not allow for co-delivery of heterologous DNA, heterologous RNA and heterologous protein into mammalian cells. For delivery of heterologous protein into mammalian cells, Baculovirus 2

Display is limited by the covalent fusion of heterologous proteins to viral envelope proteins on LU103239 the solvent facing side of the baculovirus particle. For delivery of heterologous protein into mammalian cells, Baculovirus Display thereby does not enable release of untethered heterologous proteins into the cytoplasm or nuclei of transduced cells. Baculovirus Display additionally exposes heterologous proteins fused to viral envelope proteins to the chemically non-reductive environment of insect cell media during production in insect cells, a process which takes days to weeks, and thereby is incompatible with oxidation-sensitive proteins.

The “BacMam” technology is a modification of the above described baculovirus expression systems in insect cells. BacMam uses the baculovirus particle as a vehicle to deliver heterologous DNA expression cassettes into mammalian cells. In contrast to standard baculovirus expression systems for expression in insect cells, with BacMam systems the heterologous expression cassettes are equipped with promoters which fire in mammalian cells. Upon transduction of mammalian cells with such designed baculovirus particles, heterologous DNA expression cassettes are transcribed, and recombinant protein is then produced inside the transduced mammalian cell (Boyce and Bucher 1996, Kost et al. 2005,

Mansouri and Berger 2018). The BacMam system does not transcribe the user's heterologous recombinant protein in insect cells and does not use the heavily transcribed baculovirus very late promoters for user-selected recombinant protein expression; rather it typically employs high yield mammalian or viral promoters, because baculovirus very late promoters are not transcribed in mammalian cells. The BacMam system thereby essentially uses insect cells as a production platform for creation of the baculovirus particles which carry heterologous DNA expression cassettes under the control of mammalian promoters for transduction of mammalian cells, followed by expression of heterologous proteins in said transduced mammalian cells. BacMam expression can thereby be thought of a 2-stage process- i) a virus production phase in insect cells: leading to assembly of baculoviral particles which contain genomes encoding heterologous expression cassettes driven by mammalian promoters; ii) transduction of mammalian cells with said baculoviral particles, whose heterologous expression cassettes are then transcribed inside the mammalian cell.

Wild type baculoviruses transduce mammalian cells with very low efficiency, and improved entry into vertebrate cells has been achieved by modification of native baculovirus envelope proteins and/or incorporation of exogenous envelope proteins (pseudotyping), particularly

Vesicular Stomatitis Virus G glycoprotein (VSV-G) (Barsoum et al. 1997, Kitagawa et al. 2005, Kaikkonen et al. 2006). VSV-G pseudotyping functions mechanistically by enabling the baculovirus to enter cells via low density lipoprotein (LDL) receptors, which are present in high amounts on the surface of most mammalian cell types (Finkelshtein et al., 2013). The 3

VSV-G pseudotyping strategy was therefore rapidly adapted by BacMam systems to enable LU1032539 more efficient entry of baculovirus into diverse mammalian cell types. A detailed investigation into the effect of VSV-G pseudotyping of baculovirus particle structure and function was carried out by Barsoum et al. (1997), and Mangor et al. (2001). These studies revealed that baculovirus particles pseudotyped with VSV-G are heterogeneous in structure, with some irregular shaped, larger diameter/volume viral particles present, presumably due to modified physicochemical properties of their VSV-G envelope. Although not stated by Barsoum et al. (1997), Mangor et al. (2001), or any other disclosure to our knowledge, we postulated that these larger volume virus particles caused by VSV-G pseudotyping might carry inside their irregular-shaped envelope an encapsulated sample of the cytoplasm from the insect host cell which produced the virus. By utilizing the MultiBacMam™ (Geneva Biotech) multigene

BacMam delivery system (Mansouri et al. 2016) we demonstrate in the present disclosure that heterologous protein and/or heterologous RNA produced in insect cells is indeed incorporated into VSV-G pseudotyped baculovirus particles.

It is one object of this invention to provide a pseudotyped baculovirus system for BacMam which uses the production phase in insect cells to load baculoviral particles not only with heterologous DNA expression cassette(s) as is the case with prior art BacMam systems, but in addition, to load the pseudotyped baculoviral particles with heterologous protein cargos and/or heterologous RNA cargos which were produced by user designed heterologous expression cassettes, that are expressed in insect cells. By subsequently transducing mammalian cells with said baculovirus particles, this allows for delivery of heterologous DNA, plus heterologous RNA and/or heterologous protein into said mammalian cells. In summary, prior art BacMam systems allow for delivery of heterologous DNA into mammalian cells, while no prior art BacMam systems allow for delivery of heterologous DNA, plus heterologous RNA and/or heterologous protein into mammalian cells.

Prior art baculovirus systems efficiently transduce only a limited subset of mammalian cell types. The factors affecting host cell permissiveness to baculovirus transduction are still largely unknown (Turkki et al., 2013). Transduced baculoviral DNA is not functional in a mammalian cell until it is transported into the nucleus of the cell, and baculovirus DNA is not transported to the nuclei of many industrially important mammalian cell types following transduction, and instead remains in the cytoplasm following endosomal escape of the viral capsid (van Loo et al., 2001). Therefore, the RNA and/or protein delivery function of the herein described invention is anticipated to overcome this limitation of prior art baculovirus systems for several industrially important mammalian cell types since RNA and protein can 4 be functional immediately upon release into the cytoplasm of the transduced mammalian LU103239 cells.

It is therefore a second object of this invention to provide a pseudotyped baculovirus system which broadens the list of mammalian cells types, tissues and organisms that baculovirus can effectively modulate by adding protein and RNA transduction functions on top of the existing DNA transduction functions of prior art BacMam systems.

Delivery of DNA expression cassettes into mammalian cells represents a serious safety issue for many medical applications, if said DNA encodes an element that can contribute to potential safety risks, e.g. via ongoing expression of the cassette over long periods of time.

For example, oncogenic transgenes or genome destabilizing agents such as DNA cleaving enzymes both represent safety issues if permanently integrated into the host mammalian cell genome, as they can both lead to cancer. The fields of gene editing and gene therapy both utilize such types of potentially dangerous DNA cargos, and both retroviruses and adeno- associated virus (rAAV) vectors integrate into the chromosome of cells that have been transduced with these vectors. Due to a lack of viable alternatives, retroviruses and rAAV vectors are nonetheless utilized in gene editing and gene therapy applications. In contrast to the genomic integration observed with retroviruses and rAAV vectors following mammalian cell transduction, baculovirus DNA either is stranded in the cytoplasm and remains transcriptionally inactive or is transported to the nuclei and persists in the cell as a nuclear episome whose copy number decreases with subsequent cell divisions (Hofmann et al. 1995,

Pieroni et al., 2001; Shoji et al., 1997). State of the art BacMam systems can thereby be considered as transient mammalian transgene expression systems.

It is a third object of this invention to provide a baculovirus system which provides an alternative, safer transduction system for mammalian cells, tissues or organisms in cases where permanent genome integration can potentially cause safety issues.

By utilizing the MultiBacMam™ (Geneva Biotech) multigene BacMam delivery system,

Mansouri et al. (2016) demonstrated that permanent modification of the mammalian genome via CRISPR/Cas9 could be carried out where both i) the components necessary to modify the genome, and ii) the heterologous DNA code to be integrated into the genome was encoded in one baculovirus genome. In Mansouri et al. (2016), Cas9 and guide RNAs were encoded in the baculovirus DNAs under promoters active in the target mammalian cells. This setup precisely creates the safety issue described above, where DNA expression cassettes expressing Cas9 and guide RNAs could become permanently integrated into the host 5 mammalian cell genome, which creates an actual (and regulatory) risk that the encoded DNA LU103239 cleaving enzymes would be permanently produced, destabilizing the genome and potentially leading to cancer.

Itis a fourth object of this invention to provide a baculovirus system where components necessary to modify the genome (e.g. Cas9 and guide RNAs, or Transposases) are delivered into the target mammalian cell as RNA, protein or protein-RNA complexes, rather than DNA that encodes said RNA, protein or protein-RNA complexes so as to eliminate the potential safety issue of stable transgene expression from chromosomally integrated DNA cleaving enzymes. RNA, protein or protein-RNA complexes have a very limited half-life in cells, and thereby delivering these components into mammalian cells provides significant safety advantages relative to heterologous DNA cargos which then produce said RNA, protein or protein-RNA complexes inside the transduced mammalian cells, possibly permanently upon genome integration.

The baculovirus system of the disclosed invention will be applicable to enable many distinct processes in mammalian cells, such as modifying intracellular organization, metabolism, responsiveness, signaling, movement, reproduction, or triggering death. The system is anticipated to have numerous important applications in the medical and biotech communities, including in gene therapy, cell therapy, immunotherapy, drug discovery, diagnostics, and in delivery of systems for the purpose of gene editing, RNA interference, and iPS reprogramming. Many of these applications will require the manufacturing of the baculovirus system of the disclosed invention at industrial scale and GMP (Good Manufacturing Practice) quality standard.

It is therefore a fifth object of this invention to provide streamlined production and scale-up methods adaptable to industrial scale production, and DNA component compositions compatible with regulatory compliance. Said streamlined baculovirus production methods, and DNA component compositions are therefore also disclosed in the invention.

Summary of the invention

The above and other objects of the invention are attained by the provision of the present invention as defined in the claims and further disclosed in the present description as well as the drawings. 6

By virtue of the baculovirus vector system of the present invention, it is made feasible to LU103239 assemble pseudotyped baculoviral particles in insect cells containing a cargo comprising heterologous DNA plus heterologous gene product(s) such as heterologous RNA and/or heterologous protein. This cargo of heterologous DNA plus heterologous gene product(s) such as heterologous RNA and/or heterologous protein is all co-produced in an insect cell, and thereby allows for the multicomponent cargo to be carried by an individual virus particle.

The result of this co-production in insect cells is a baculovirus particle carrying a combined payload of heterologous DNA, plus heterologous gene product(s) such as heterologous RNA and/or heterologous protein together in a baculovirus, preferably in the interior of the baculovirus, more preferably not covalently bound to a part of the baculovirus, which is coated with (“pseudotyped”) particular heterologous proteins which enable efficient cell entry (transduction) of a broad range of mammalian cell types, including primary cells, tissues, and whole mammalian organisms. The baculovirus vector system of the present invention is broadly applicable across biomedical applications including gene therapy, cell therapy, immunotherapy, drug discovery, synthetic biology and others.

In particular, the present invention provides, under a first aspect, a baculovirus vector system comprising one or more recombinant vectors comprising the following elements: (1) a baculovirus genome sufficient for producing baculovirus particles upon transfection in susceptible insect cells; (2) at least one heterologous expression cassette À containing a promoter and an open reading frame coding for at least one modified viral envelope or coat, respectively, protein, for example, V-VSG, the expression cassette enabling the expression of the at least one modified viral envelope protein in insect cells and incorporation of the at least one modified viral envelope protein into the baculovirus particle upon virus production in insect cells; and (3) at least one heterologous expression cassette B containing a promoter and a nucleotide sequence coding for at least one heterologous gene product, the expression cassette enabling the expression of said gene product(s) in insect cells and incorporation of the gene product(s) into the baculovirus particle upon virus production in insect cells with the proviso that, if the gene product is a heterologous polypeptide it is an essentially non-fluorescent polypeptide, with the proviso that the heterologous polypeptide is optionally fused to a fluorescent label. ltis preferred according to the invention that the one or more gene products of expression cassette(s) B is/are incorporated into the interior of the baculovirus particle. Internal incorporation of the one or more expression cassettes B is beneficial because the one or 7 gene products is/are present in a chemically protected environment inside the baculovirus | LU103239 particle, e.g. the interior or the virus particle provides a reducing which is important for folding and retaining function of heterologous polypeptides which typically lose their stability and functionality in a non-reducing environment. More preferably, the one or more gene products of expression cassette(s) B are incorporated into the baculovirus particle in a non-covalent fashion, i.e. not covalently bound to another part of the baculovirus particle.

For increasing the of integration of the one or more heterologous gene products of expression cassette B it is preferred to provide a non-covalent interaction of a non-covalent binding pair into the expression system of the invention. In certain embodiments, one or both of elements (1) and (2) may comprise nucleotide sequences encoding a partner of a non- covalent binding pair which may be arranged such that it is fused to a component of the interior of the baculovirus particle, and the other partner of the non-covalent binding pair is incorporated into element (3), e.g. the gene product(s) of expression cassette B include sequences encoding the other partner of the non-covalent binding pair, preferably fused to the one or more gene products. Non-limiting useful examples of such non-covalent binding partner are dimerization domains such as leucine zipper domains or helix-loop-helix domains, or other systems of non-covalent domains include peptide binding proteins together with their substrate peptides, antigen-antibody interaction partners, protein-adaptor protein interaction partners, protein-chaperone interaction partners and protein-snare complex formation protein-scaffold protein interaction partners.

Elements (1) to (3) may be contained in one or more vectors. For example, all three elements (1) to (3) may be present in a single vector. In other embodiments, the elements can be contained in two vectors. For example, elements (1) may be contained in a first vector, and elements (2) and (3) are contained in a second vector. In other embodiments, elements (1) and (2) are contained in first vector, and element (3) is contained in a second vector. Alternatively, elements (1) and (3) may be included in a first vector and element (2) may be included in a second vector. In further embodiments of the invention, each of elements (1) to (3) is contained in a separate vector, i.e, the baculovirus vector system comprises three vectors.

In preferred embodiments of the invention, the vector system comprises a vector, typically a bacmid, containing element (1), and one vector containing elements (2) and (3), or in other preferred embodiments, the vector system comprises a vector, typically a bacmid, containing element (1), a second vector containing element (2) and a third vector containing element (3). The vector(s) containing element (2) and/or (3) are often referred to as “transfer vectors”. 8

The gene product of expression cassette B may be any gene product expressible from said promoter in insect cells.

Preferably, the at least one heterologous expression cassette B contains a promotor and (i) an open reading frame coding for at least one heterologous essentially non-fluorescent polypeptide, optionally fused to a fluorescent label, and/or (ii) a sequence coding for at least one heterologous RNA transcribed by the promoter. In certain embodiments of the invention an expression cassette B is denoted as “expression cassette B1” in case the gene product is a heterologous polypeptide as defined above, and an expression cassette B is denoted as “expression cassette B2” in case the gene product is an (untranslated) RNA.

Thus, in one embodiment, the gene product is a heterologous RNA including, but not limited to, MRNA, tRNA, snRNA and snoRNA.

In other embodiments, the gene product is a heterologous polypeptide, preferably protein, provided that it is not a self-fluorescent polypeptide, i.e. essentially non-fluorescent polypeptide. “Essentially non-fluorescent polypeptide” means according to the invention that the heterologous polypeptide as a gene product of element (3) does not in itself, i.e. as long as it does not include (such as typically coupled to) a fluorescent label or entity, respectively, emit fluorescence at a reasonably detectable level. “Reasonably detectable level of fluorescence” means in the context of the invention that the fluorescence is lower than the detection level of a commercially available photometer or of a fluorescence microscope. The limit of detection (LOD) is typically defined as the sample concentration at which the signal is equal to 3x the noise level of the device and is thus limited by system noise. Detection limits for commercially available photometers can range from nanomolar to picomolar concentrations for certain assays, but this can vary based on the specific design and application of the photometer. Detection limits for fluorescence microscopy can range from low nanomolar to high picomolar concentrations, depending on the specific microscope setup and the fluorophores used.

In certain embodiments, expression cassette B may comprise a nucleotide sequence which provides for expression of both an, typically non-coding, RNA and a heterologous polypeptide.

In preferred embodiments of the invention, the system further comprises at least one expression cassette C, optionally contained in the baculovirus genome (1), containing a 9 promoter and nucleotide sequence coding for at least one heterologous gene product, the ~~ LU103239 expression cassette enabling the expression and/or transcription of said gene product in mammalian cells.

Preferably, the at least one expression cassette C contains a promotor and (a) an open reading frame coding for at least one heterologous polypeptide and/or (b) a sequence coding for at least one heterologous RNA transcribed by the promoter, wherein the heterologous polypeptide and the heterologous RNA is/are expressed or transcribed, respectively, in mammalian cells. It is to be understood that a heterologous polypeptide encoded by the open reading frame of expression cassette C may also be, unlike the heterologous polypeptide of expression cassette B, a fluorescent protein. In certain embodiments of the invention an expression cassette B is denoted as “expression cassette C1” in case the gene product is a polypeptide as defined above, and an expression cassette B is denoted as “expression cassette C2” in case the gene product is an (untranslated) RNA.

Preferably, the heterologous polypeptide and/or the heterologous RNA (i.e. the gene product(s) of expression cassette(s) B and/or C) is/are modulators of a physiological process selected from the group consisting of intracellular organization, metabolism, responsiveness, immune system processes, cell signaling, movement, genome modifications, reproduction, or (preferably cell) death.

In preferred embodiments, the term metabolism includes a cellular process selected from intracellular trafficking, RNA transcription, protein translation and cellular energy levels.

The term “immune system process” preferably includes, but is not limited to, innate, cellular and adaptive immune system processes.

The term “genome modification” preferably includes, but is not limited to, base substitutions, base deletions, base insertions, and epigenetic modifications of host cell DNA.

Preferred viral envelope proteins include, but are not limited to, VSV-G, HIV gp120, baboon envelope protein, and modified versions of baculovirus envelope protein GP64.

Preferably, the VSV-G comprises, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 3 or a transcriptionally functional homolog thereof. 10

It is further preferred that the transcriptionally functional homolog of VSV-G has a sequence LU103239 identity at least 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 % to SEQ ID NO: 3.

In certain preferred embodiments of the invention, element (2) of the baculovirus vector system comprises at least one further expression cassette containing a promoter and an open reading frame coding for an additional modified viral envelope protein comprising an immune complement system inhibitor, wherein the additional modified viral envelope protein is expressed in insect cells and incorporated into the baculovirus particle upon virus production in insect cells.

Preferred immune complement system inhibitors for use in the inventions include, but are not limited to, decay-accelerating factor (DAF), factor H (FH)-like protein-1 (FHL-1), C4b-binding protein (C4BP), and membrane cofactor protein (MCP). ltis further preferred according to the invention that the immune complement system inhibitor is fused to a moiety selected from the group consisting of the baculovirus envelope protein

GP64 and the membrane anchor of the vesicular stomatitis virus-G (VSV-G) protein.

Preferably, protein GP64 comprises, essentially consist of or consists of the amino acid sequence of SEQ ID NO: 13 or a transcriptionally functional homolog thereof.

It is further preferred that the transcriptionally functional homolog of GP64 has a sequence identity at least 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 % to SEQ ID NO: 13.

Preferably, the membrane anchor comprises, essentially consists of or consists of the amino acid sequence of SEQ ID NO: 14 or a transcriptionally functional homolog thereof. It is further preferred that the transcriptionally functional homolog of the membrane anchor according to SEQ ID NO: 14 has a sequence identity at least 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 % to.

In preferred embodiments of the invention, the complement system inhibitor fused to GP64 is a DAF-GP64 fusion protein.

In certain preferred embodiments of the invention, the complement system inhibitor fused to the membrane anchor of VSV-G is a DAF-VSV-G membrane anchor fusion protein. 11

According to certain preferred embodiments of the invention, heterologous RNA produced by LU103239 expression cassette(s) B comprises sequence elements that enable the RNA to be translated in mammalian cells. For example, the the sequence elements enabling the RNA to be translated in mammalian cells may comprise at least one internal ribosomal entry site (IRS).

In preferred embodiments of the invention, expression cassette B codes for a heterologous polypeptide selected from the group consisting of apoptosis factors, iPSC reprogramming factors, tumor suppressor proteins, tumor suppressor /peptides, and vaccines.

Preferred apoptosis factors include, but are not limited to, Apoptosis Inducing Factor, caspase-3, caspase-8, and caspase-9.

Preferred iPSC reprogramming factors include, but are not limited to, Oct4, Sox2, cMyc, KIf4,

LIN28A, NANOG, L-MYC, GLIS1, ESRRB, UTF1, NR5A2, SALL4, PRDM14, DPPA2,

DPPA4, TDGF1, RARG, MBD3, NR2F2, ZIC3, ASCI1, LMX1A, MYT1L, BM2 AND NURR1.

Preferred tumor suppressor proteins/peptides include, but are not limited to, p53, pRB,

BCL2, SWI/SNF, pVHL, APC, CD95, ST5, YPEL3, ST7, and ST14, p16, BRCA2, APC and

NBD peptides.

A particularly preferred tumor suppressor for expression using the vector system according to the invention is p53. More, preferably, p53 comprises, essentially consists of or consists of the amino acid sequence of SEQ ID NO: 10 or a transcriptionally functional homologue thereof. It is further preferred that the transcriptionally functional homologue of SEQ ID NO: 10 has a sequence identity at least 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 % to SEQ ID

NO: 10.

Preferred vaccines for expression using the vector system of the invention include, but are not limited to protein vaccines, peptide vaccines, and RNA vaccines. In preferred embodiments of the invention the vaccine may selected from live-attenuated vaccines, inactivated vaccines, subunit, recombinant, polysaccharide, conjugate vaccines, and toxoid vaccines.

According to preferred embodiments of the invention, wherein the expression cassette(s) B and/or C is/are coding for an RNA, the RNA may preferably be selected from siRNAs, dsRNAs, shRNAs, miRNAs, ribozymes, dsRNA, shRNAs and miRNAs. 12

Preferred heterologous elements included in or encoded as heterologous gene products by, LU103239 respectively, expression cassette(s) B and/or C is/are selected from a DNA endonuclease protein, a transposon, a DNA base editor, a prime editor, one or more or all elements such as one or more protein and/or one or more RNAs of a transposon-encoded CRISPR-Cas system, a Cas-Reverse Transcriptase fusion protein and a transposase protein. It is to be understood that expression cassettes B and/or C may encode one or more than one heterologous peptide, polypeptide and/or proteins and/or include one or more than one sequence elements such as one or more of the preferred examples outlined above.

Preferred endonuclease proteins include, but are not limited to, RNA-guided nucleases,

TALENS, Zn Finger Nucleases, and homing endonucleases.

Preferred elements (one or more thereof) of transposon-encoded CRISPR-Cas systems include, but are not limited to, Th6677-based systems (Klompe et al., 2019), preferably those including the DNA-targeting complex Cascade and the transposition protein TniQ.

Preferred RNA-guided nucleases include, but are not limited tom, RNA-guided nucleases form CRISPR-Cas system types |, II, II, IV, V and//or VI and DNA base editors.

Particularly preferred RNA-guided nucleases include, but are not limited to, Cas9, dCas9,

SpCas9, Cas9 10a nickase, Cas 12b, PE1, PE2 and Cpft1.

Preferably, Cas9 comprises, essentially consists of or consists of the amino acid sequence of

SEQ ID NO: 6 or a transcriptionally functional homologue thereof. It is further preferred that the the transcriptionally functionally homologue of SEQ ID NO: 6 has a sequence identity at least 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 % to SEQ ID NO: 6.

Preferably, dCas9 comprises, essentially consists of or consists of the amino acid sequence of SEQ ID NO: 7 or a transcriptionally functional homologue thereof. It is further preferred that the transcriptionally functionally homologue of SEQ ID NO: 7 has a sequence identity at least 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 % to SEQ ID NO: 7.

Preferably, PE1 comprises, essentially consists of or consists of the amino acid sequence of

SEQ ID NO: 8 or a transcriptionally functional homologue thereof. It is further preferred that the transcriptionally functionally homologue of SEQ ID NO: 8 has a sequence identity at least 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 % to SEQ ID NO: 8. 13

Preferably, PE2 comprises, essentially consists of or consists of the amino acid sequence of LU103239

SEQ ID NO: 9 or a transcriptionally functional homologue thereof. It is further preferred that the transcriptionally functionally homologue of SEQ ID NO: 9 has a sequence identity at least 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 % to SEQ ID NO: 9.

In preferred embodiments oof the invention wherein the vector system is arranged to provide expression of one or more elements of CRIPR-Cas systems, it is further preferred that the vector system comprises at least one additional expression cassette B and/or C coding for at least one guide RNA, preferably a single guide RNA (sgRNA) and/or a prime editing guide

RNA (pegRNA).

Preferred transposons for inclusion in expression cassette B and/or C include retrotransposons and DNA transposons. Particularly preferred transposons include, but are not limited to, Transposon TN3, Transposon TN10, Transposon Tn6677, and Transposon

TN7. More preferably, Transposon TN7 comprises or consists of genes tnsA, tnsB, tnsC, and tnsD. In preferred embodiments, Transposon TN7 additionally encodes an RNA-guided nuclease.

According to preferred embodiments of the invention, at least one expression cassette C as defined as defined herein comprises one or more recognition motifs of a protein preferably selected from a DNA endonuclease protein, a DNA base editor, a prime editor, a transposon- encoded CRISPR-Cas system, preferably one or more or all elements thereof such as one or more protein and/or one or more RNA components thereof, a Cas-Reverse Transcriptase fusion protein and a transposase protein, enabling said one or more recognition motifs being bound, modified and//or cleaved by said protein.

In preferred embodiments of the invention, the DNA endonuclease protein, the DNA base editor, the prime editor, the transposon-encoded CRISPR-Cas system, preferably one or more or all elements thereof such as one or more protein and/or one or more RNA components thereof, the Cas-Reverse Transcriptase fusion protein and/or the transposase protein as described above, preferably one or more of the preferred embodiments outlined before, are selected such that said DNA endonuclease protein, DNA base editor, prime editor, transposon-encoded CRISPR-Cas system, preferably one or more or all elements thereof such as one or more protein and/or one or more RNA components thereof, Cas-

Reverse Transcriptase fusion protein and/or transposase protein, respectively, bind(s), modifies/modify and/or cleave(s) one or more recognition motifs present in a mammalian cell. 14

In further preferred embodiments of the invention expression cassette C is selected such that LU103239 it becomes permanently integrated into a mammalian cell by an endogenous mammalian

DNA repair system and/or by an DNA endonuclease protein, DNA base editor, prime editor, transposon-encoded CRISPR-Cas system, preferably comprising one or more or all elements thereof such as one or more protein and/or one or more RNA components thereof,

Cas-Reverse Transcriptase fusion protein and/or transposase protein as outlined above.

In preferred embodiments of the invention the total combined size of heterologous expression cassettes A, B, and, optionally, C is more than 7,500 bps, preferably more than 10,000 bps, more preferably 15,000 bps, even more preferably more than 20,000 bp.

Preferred promoters for use in the expression cassettes, in particular expression cassette(s)

B and/or C, include, but are not limited to, Polyhedrin promotors and p10 promoters.

More preferably, the Polyhedrin promoter comprises, essentially consists of or consists of the nucleotide sequence of SEQ ID NO: 1. It is further preferred that the p10 promoter comprises, essentially consists of or consists of the nucleotide sequence of SEQ ID NO: 2.

In preferred embodiments of the invention the baculovirus genome is derived from a nuclear polyhedrosis virus (NPV). Preferred NPV baculovirus genomes for use in the present invention are or are derived from the multiple nucleocapsids per envelope (MNPV) subgenera of the NPV genera of the Eubaculovirinae subfamily (occluded Baculoviruses) of the Baculoviridae family of insect viruses. The most preferred baculovirus genome for use in the invention is the genome of or derived from the genome of, respectively, Autographa californica nuclear polyhedrosis virus (AcMNPV).

In preferred embodiments of the invention the expression cassette(s) A and/or B is/are contained in a transfer vector suitable for fusion with genetically modified baculovirus DNA, in a modified baculovirus DNA, in a separate chromosomal DNA within insect cells infected with a baculovirus, or in a non-chromosomal DNA within insect cells infected with a baculovirus.

The present invention is further directed to a composite baculovirus DNA comprising the baculovirus vector system of the invention or comprising the elements of the baculovirus vector system, respectively.

The invention further provides a host cell comprising the baculovirus vector system or the composite baculovirus DNA. Preferred host cells of the invention include, but are not limited 15 to, bacteria, yeast and insect cells. Especially preferred insect cells comprise the composite LU103239 baculovirus DNA of the invention. Preferred insect cells include, but are not limited to, insect cells derived from Spodoptera frugiperda, Trichoplusia ni, Plutella xylostella, Manduca sexta, and Mamestra brassicae.

An especially preferred insect cell for use as a host cell of the invention is an IPLB-SF21AE cell or its clonal isolate Sf9.

The present invention also provides a cell culture comprising a host cell of the invention in a cell culture medium adapted for storage and/or propagation of the host cell.

Further subject matter of the invention is a recombinant baculovirus particle comprising the composite baculovirus DNA of the invention.

The present invention is also directed to a recombinant baculovirus particle comprising element (1) as defined herein, at least one modified viral envelope protein expressed from the heterologous expression cassette A as defined herein, at least one heterologous gene product expressed from the at least one heterologous expression cassette B as defined herein, and, optionally, at least one heterologous gene product expressed from the at least one heterologous expression cassette C as defined herein.

Further subject matter of the invention is a pharmaceutical composition comprising a host cell of the invention or cell culture according to the invention or a recombinant baculovirus particle of the invention. Preferably, the pharmaceutical composition comprises in addition to the Active Pharmaceutical Ingredient (such as the host cell, cell culture, baculovirus particle) at least one pharmaceutically acceptable excipient and/or carrier and/or diluent.

The baculovirus vector system and/or the composite baculovirus DNA can successfully be used in a variety of applications involving the transfer of heterologous gene products such as heterologous peptides, polypeptides, proteins, polynucleotides, preferably RNA, but also

DNA into mammalian cells. Preferably, the above items of the invention, preferably the baculovirus vector system and/or the composite baculovirus DNA, are used in gene therapy, gene editing, gene engineering, cell therapy, immunotherapy, delivery of RNA interference systems, delivery of apoptosis factors, iPS reprogramming, synthetic biology, production of protein complexes, modification of cell signaling, drug discovery, diagnostics, and vaccines. 16

Further subject matter of the invention is a method for delivering DNA together with one or LU103239 more heterologous gene products such as preferably heterologous peptides, polypeptides, proteins, polynucleotides, preferably RNA, but also DNA, into a mammalian cell and/or a mammalian tissue and/or a mammalian organ and/or mammalian organism comprising the steps of: (a) introducing the baculovirus vector system as disclosed herein and/or the composite baculovirus DNA as disclosed into insect cells, preferably insect cells as outlined above; (b) culturing said insect cell under conditions allowing the expression of the gene product of expression cassette B and, optionally, expression cassette C, and allowing the production of recombinant baculovirus particles; (c) harvesting the baculovirus particles produced in step (b); and (d) transducing the mammalian cell, tissue, organ, and/or organism with the baculovirus particles harvested in step (c).

In preferred embodiments of this method the culture conditions are maintained until the number of insect cells is between about 102 and 10" and/or until the number of baculovirus particles is between 10% and 10".

Preferred mammalian cell types include, but are not limited to, primary cells, immortal cell lines, cells being part of a tissue and a cell being part of an organ from a living mammal.

Preferred primary cells include, but are not limited to, immune cells, neuronal cells, adipose cells, bladder cells, blood vessel cells, cardiac cells, cartilage cells, bone cells, bone marrow cells, bronchial/tracheal cells, cardiac cells, colon cells, dermal cells, epidermal cells, esophagus cells, gallbladder cells, gastrointestinal cells, hepatic cells, keratinocytes, lung cells, lymphatic cells, mammary cells, ocular cells, pancreatic cells, iPS cells, fertilized oocytes and unfertilized oocytes.

Preferred primary immune cells include, but are not limited to, B cells, T cells, Dendritic cells,

TILs, activated T cells, and resting T cells.

Preferred organs include, but or not limited to, arteries, bones, bone marrow, brain, gallbladder, heart, intestines, kidneys, larynx, liver, lung, lymph nodes, muscles, ovary, pharynx, placenta, prostate, skin, spleen, stomach, thyroid gland, urethra, urinary bladder, and veins. 17

The Figures show: LU103239

Fig. 1: refers to Example 1 below and shows in Schematic Drawings components of a preferred embodiment of the baculovirus vector system of the invention and the composition of baculovirus particles after production in insect cells. Baculovirus

Expression Vector System (BEVS) delivery of a red fluorescent protein, mCherry, produced in insect cells, into mammalian (CHO) cells together with heterologous expression cassette encoding a green fluorescent protein (eGFP) under the control of a hPGK promoter that fires in the mammalian cells. Top: BEVS DNA components

A) B) and C) are shown schematically. A) comprises the VSV-G coding sequence under the control of the baculovirus polh promoter; B) comprises the mCherry coding sequence under the control of the baculovirus p10 promoter; C) comprises the eGFP coding sequence under the control of the mammalian hPGK promoter. Middle: baculovirus particle composition after production in insect cells is shown schematically. VSV-G glycoprotein produced in insect cells is incorporated into the viral envelope of the baculoviral particle to facilitate transduction into mammalian cells. Protein mCherry produced in insect cells is incorporated into the baculoviral particle for delivery into the mammalian cells following transduction. Bottom:

Mammalian (CHO) cell following transduction with baculovirus particle. mCherry protein is delivered into the CHO cell by the baculoviral particle, along with DNA coding for eGFP under control of hPGK promoter which is transcribed and then translated in the transduced mammalian (CHO) cells.

Fig. 2: is a schematic representative of Transfer vector pEXAMPLE1.1. DNA components B) and C) for Example 1 are shown schematically in plasmid pFL (Fitzgerald et al. 2006). For component B) the mCherry coding sequence under the p10 promoter is indicated (right), and for component C) the eGFP coding sequence under the hPGK promoter is indicated (top).

Fig. 3: shows a schematic representation of the strategy to introduce pEXAMPLE1.1 into the baculovirus genome DH10EMBacVSV. Right: plasmid pEXAMPLE1.1 is described schematically. Left: Shown is a schematic drawing of the DH10EMBacVSV baculovirus bacmid shuttle vector genome indicating how the VSV-G expression cassette under control of the baculovirus very late p10 promoter was introduced into the DH10MultiBac genome (Mansouri et al. 2016). Indicated with a dashed cross is the use of Tn7 transposition to introduce plasmid pEXAMPLE1.1 into the

DH10EMBacVSV genome (Fitzgerald et al. 2006). 18

Fig. 4A: shows the flow cytometry method used to determine and quantify the presence of fluorescent proteins inside CHO cells. CHO cells cultured in CD OptiCHO™ medium (ThermoFischer) were seeded in 24 well plates (0.5 ml per well) and these adherent cells incubated at 37°C, 5% CO2 for 24h. The cells were then transduced with the baculovirus of Figure 3 at multiplicity of infection (MOI) of 5.0, followed by 3h incubation at 25°C on a plate rocker at 30 motions per minute. Following the 3h incubation, the transduction suspension was quantitatively removed and replaced by fresh CHO culture media. The cells were then incubated at 37°C, 5% CO2 for a further 24h.

Analysis of cells via Fluorescence-Activated Cell Sorting (FACS) was carried out as described in (Kim et al, 2012).

The cluster corresponding to CHO cells was identified (Figure 4A, Left), and only the events falling in this region were kept for analysis. The threshold for fluorescence (Figure 4A, Right) was determined by setting the percentage of fluorescent cells in this sample identical to an untransduced sample run as a control. This value was typically slightly above zero (0.1 to 1.0% of cells) where this small percentage appears to be due to visible auto fluorescence of dying cells, or debris which auto fluorescence. In this example (Figure 4A, Right) 89.58 % of the cells were determined to be fluorescent green indicative of the presence of eGFP.

Fig. 4B: shows simultaneous delivery by a baculovirus particle of mCherry protein into CHO cells with a DNA expression cassette encoding eGFP. CHO cells cultures as described in Figure 4A were transduced with the virus described in Figure 3 at different MOIs and analyzed by FACS. FACS was used to determine the % of cells containing eGFP (left) or mCherry (right), as a function of increasing MOI. As can be seen in the figure, >80% delivery/transduction of both eGFP and mCherry can be achieved with MOIs of 5.0 and above.

Fig. 5: shows schematic representations of the baculovirus vector system DNA components and the baculovirus particle composition after their production in insect cells according to Example 2. Baculovirus Expression Vector System (BEVS) delivery of a caspase protein, iCasp9, produced in insect cells, into mammalian Jurkat cells together with heterologous expression cassette encoding a Chimeric Antigen

Receptor (CAR). Top: BEVS DNA components A) B) and C) are shown schematically.

A) comprises the VSV-G coding sequence under the control of the baculovirus polh promoter; B) comprises the iCasp9 coding sequence under the control of the 19 baculovirus p10 promoter; C) comprises the CAR coding sequence under the control LU103239 of the mammalian hPGK promoter. Middle: baculovirus particle composition after production in insect cells is shown schematically. VSV-G glycoprotein produced in insect cells is incorporated into the viral envelope of the baculoviral particle to facilitate transduction into mammalian cells. Protein iCasp9 produced in insect cells is incorporated into the baculoviral particle for delivery into the mammalian cells following transduction. Bottom: Mammalian (CHO) cell DNA and protein composition following transduction with baculovirus particle. iCasp9 protein is delivered into the

Jurkat cell by the baculoviral particle, along with DNA coding for the CAR under control of a mammalian promoter.

Fig. 6: shows a schematic representation of transfer vector pEXAMPLE2.1. DNA components B) and C) for Example 2 are shown schematically in plasmid pFL (Fitzgerald et al. 2006). For component B) the iCasp9 coding sequence under the p10 promoter is indicated (right), and for component C) the CAR coding sequence under the hPGK promoter is indicated.

Fig. 7: shows in a schematic representation the strategy to introduce pEXAMPLE2.1 into the baculovirus genome DH10EMBacVSV according to Example 2. Right: plasmid pEXAMPLE2.1 is described schematically. Left: Shown is a schematic drawing of the

DH10EMBacVSV baculovirus bacmid shuttle vector genome indicating how the VSV-

G expression cassette was introduced into the DH10MultiBac genome (Mansouri et al., 2016). Indicated with a dashed cross is the use of Tn7 transposition to introduce plasmid pEXAMPLE2.1 into the DH10EMBacVSV genome.

Fig. 8: shows simultaneous delivery by a baculovirus particle of iCasp9 protein into Jurkat cells with a DNA expression cassette encoding a CAR. Jurkat cell cultures were transduced with the virus described in Figure 7 at different MOIs and analyzed by

FACS to determine the physical presence of the CAR, and the functional activity of the iCasp9 protein inside the transduced cell. Figure 8, left, shows the % of cells expressing the CAR as a function of increasing MOI. Cell death through iCasp9 can be triggered by the small molecule chemical AP1903, where addition of AP1903 Kills cells in less than an hour. Figure 8, right, shows the % of Jurkat cells that were killed as a function of increasing MOI. As can be seen in the figure, > 80% delivery/transduction of both iCasp9 and CAR can be achieved with MOIs of 5 and above. 20

Fig. 9: shows schematic representations of the baculovirus vector system DNA components LU103239 and the baculovirus particle composition after their production in insect cells according to Example 3. Baculovirus Expression Vector System (BEVS) for co- production in insect cells of a genome editing protein, Cas9-GFP and its guide RNAs, for delivery into CHO cell together with heterologous DNA expression cassette encoding an interleukin under control of a mammalian promoter (eRFP-IL2). Top:

BEVS DNA components A) B1) B2) and C) are shown schematically. A) comprises the VSV-G coding sequence under the control of the baculovirus polh promoter; B1) comprises the Cas9-GFP coding sequence under the control of the baculovirus p10 promoter; B2) comprises guide RNAs for the Cas9-GFP also under the control of the p10 promoter; C) comprises the eRFP-IL2 coding sequence under the control of the mammalian hPGK promoter. Middle: baculovirus particle composition after production in insect cells is shown schematically. VSV-G glycoprotein produced in insect cells is incorporated into the viral envelope of the baculoviral particle to facilitate transduction into mammalian cells. Protein Cas9-GFP produced in insect cells together with their guide RNAs are together incorporated into the baculoviral particle for delivery into the mammalian cells following transduction with said baculoviral particle. Bottom: Mammalian (CHO) cell DNA and protein composition following transduction with baculovirus particle. Cas9-GFP protein is delivered into the CHO cell by the baculoviral particle, along with guide RNAs for the Cas9-GFP, along with DNA coding for eGFP under control of a mammalian promoter.

Fig. 10: shows a schematic representation of transfer vector pEXAMPLE3.1. DNA components B1) B2) and C) for Example 3 are shown schematically in plasmid pFL (Fitzgerald et al., 2008). For component B1) the Cas9-GFP coding sequence under the p10 promoter is indicated (right), and for component C) the Cas9-GFP coding sequence under the hPGK promoter is indicated. For component B2) guide RNAs are under the p10 promoter are indicated.

Fig. 11: shows in a schematic representation the strategy to introduce pEXAMPLE3.1 into the baculovirus genome DH10EMBacVSV according to Example 3.. Right: plasmid pEXAMPLES.1 is described schematically. Left: Shown is a schematic drawing of the

DH10EMBacVSV baculovirus bacmid shuttle vector genome indicating how the VSV-

G expression cassette was introduced into the DH10MultiBac genome (Mansouri et al. 2016). Indicated with a dashed cross is the use of Tn7 transposition to introduce plasmid pEXAMPLES3.1 into the DH10EMBacVSV genome. 21

Fig. 12: shows simultaneous delivery by a baculovirus particle of Cas9-GFP protein and guide LU103239

RNAs into CHO cells with a DNA expression cassette encoding eRFP-112. CHO cells cultures as described in Figure 4A were transduced with the virus described in Figure 11 at different MOIs and analyzed by FACS. FACS was used to determine the % of cells containing Cas9-GFP (top) or eRFP-II2 (middle), as a function of increasing

MOI. Bottom, shows the % of cells containing guide RNAs as measured by fluorescent in-situ hybridization to detect the guide RNAs by FACS (Young et al. 2020). As can be seen in the figure, >70% delivery/transduction of Cas9-GFP, eRFP- [12 and guide RNAs can be achieved with MOIs of 5.0 and above.

Detailed Description of the Invention

Provided herein is a baculovirus expression system as defined above. In one embodiment of this aspect, the invention provides insect cells comprising the baculovirus expression system according to the invention. Any of such embodiments of this aspect are useful for introducing DNA and heterologous gene products such as heterologous RNA and/or heterologous polypeptides in mammalian cells.

While not being bound to any theory, the present invention is at least in part based on the realization that during their generation in insect cells baculovirus particles enclose a part of the cytoplasm such that they take up cytoplasmic components present in the cytoplasm of that insect cell, and the cytoplasmic components are transported out of the insect cell when the baculovirus particles leave the cells. Thus, when heterologous gene products such as heterologous polypeptides and/or RNA, are produced in such insect cells e.g., from expression cassette(s) B and, optionally,

C contained in composite baculoviral DNA and/or from said expression cassette(s) B and, optionally, C present in one or more separate vectors in the insect cells together with the baculovirus particles (since said expression cassettes preferably comprise appropriate baculoviral promotors that fire in insect cells) the heterologous gene product(s) are contained in the cytoplasm of the insect cell and become cargo material within the generated baculovirus particles. The produced baculovirus particles containing the desired cargo can then be used, by appropriate pseudo- typing, to transduce target mammalian cells and release the cargo taken from the insect cells into the target cell following successful transduction of said mammalian cells. 22

According to the invention, a "heterologous gene product” is a product obtained by transcription (heterologous RNA) and, optionally, translation (heterologous polypeptide) encoded by a gene wherein the gene product, with reference to the gene product(s) of expression cassettes B and, optionally, C, is/are not a natural gene product of the target cell. Often, the heterologous gene product(s) is/are also heterologous with respect to the insect cells in which the baculovirus particles and the heterologous gene product(s) is/are produced. However, in certain embodiments of the invention, it is also possible according to the invention that the gene product(s) is/are a natural component of the insect cell where production of the baculovirus particles occurs. As noted above, i some instances in the present disclosure, the term “expression cassette B2” may be used to denote an expression cassette B coding for an (untranslated) RNA, and likewise the term “expression cassette C2” may be used in the present disclosure for an expression cassette C coding for an (untranslated) RNA.

As used herein “baculovirus vector system” refers to a system comprising elements (1) to (3) as defined and described herein enabling the production of baculoviruses expressing one or more heterologous gene products such as a heterologous polypeptide as defined above and/or heterologous RNA, in insect cells wherein the baculovirus particles formed in the insect cells comprises said heterologous gene products. Due to element (2) the baculovirus further comprises a modified envelope protein preferably enabling uptake of the baculovirus particles produced in insect cells by non-insect cells such as mammalian cells. Accordingly, transduction of cells comprising a protein such as a receptor to which the modified envelope protein present on the surface of the baculovirus can bind leads to uptake of the contents of the baculovirus components including the heterologous gene product(s) by the cell comprising the protein, preferably a receptor, to which the modified envelope protein of the baculovirus particle binds. Thus, the baculovirus particles carrying the heterologous gene product(s) can be used to introduce the heterologous gene product(s) expressed from expression cassettes B and, optionally, C into target cells susceptible totransduction by the baculovirus particle displaying on its surface the modified envelope protein expressed from expression cassette A of element (2) of the baculovirus vector system according to the invention. Preferred target cells are 23 mammalian cells. In that case expression cassette A of element (2) typically encodes LU103239 a VSV-G protein or transcriptionally functionally homolog thereof, typically as further defined below.

Referring to element (1) of the baculovirus vector system of the invention comprises all baculovirus DNA elements necessary for production of baculovirus particles in susceptible insect cells and recombinant protein expression in insect cells. Thus, such a system may comprise a transfer vector, a modified baculovirus DNA and optionally a helper plasmid or a modified insect cell, wherein the transfer vector can be fused to the modified baculovirus DNA to form a composite baculovirus. The composite baculovirus DNA generally comprises all components of the baculovirus expression system. In certain embodiments however, the composite baculovirus

DNA may lack one or other such components which are then comprised in a modified cell line that forms part of the expression system. Most of the presently used baculovirus vector systems are based on the sequence of Autographa californica nuclear polyhedrosis virus (ACMNPV) ((Virology 202 (2), 586-605 (1994), NCBI

Accession No.: NC_001623).

The term “virus amplification” as used herein refers to the propagation of virus and, in particular, to serially passaging a virus to scale up number of virus particles starting from a clonal virus or a clonal viral genome. Such propagation is typically conducted using, such as during culture of, insect cells.

As used herein the term “expression cassette” refers to an entity made up of a gene and the sequences controlling its expression. An expression cassette comprises at least a promoter sequence and a sequence transcribed under the control of the promoter and a 3’ untranslated region. In the case of genes encoding a protein or polypeptide, respectively, the sequence transcribed under the control of the promoter contains an open reading frame encoding the protein or polypeptide, respectively.

The expression cassette can be part of a vector DNA, plasmid DNA, bacmid DNA, genomic DNA, a virus, a cell or any other DNA/RNA or DNA/RNA containing organism. 24

Expression cassette A of the present invention contains a promoter and an open LU103239 reading frame coding for a modified viral envelope protein.

The term “promoter” as used herein is a region of DNA that facilitates the transcription of a particular gene. Promoters are typically located adjacent to the genes they regulate, on the same strand and upstream (towards the 5’ region of the sense strand).

The term “open reading frame” (ORF) as used herein is a portion of an organism's genome which contains a DNA sequence that could potentially encode a protein. In a gene, ORFs are located between the start-code sequence (initiation codon) and the stop-code sequence (termination codon).

Replication origins in baculovirus genomes are of two types, hr (homologous region), and non-hr (Wu et al., 2014). For the purposes of the present invention, a DNA integration site is considered “in proximity” (or adjacent) to a “hr” if it is situated downstream or upstream of said replication origin at a distance of preferably less than 3000 bps, more preferably less than 2000 bps, most preferably less than 1000 bps.

The term “late promoter” as used herein is a promoter used for baculovirus late gene expression, for example to express genes between about 6 and 24 hours post infection of an insect cell, such as between about 12 and 18 hours post infection. The term “very late promoter” as used herein is a promoter used for baculovirus very late gene expression, for example to express genes between about 18 and 72 hours post infection, such as between about 18 and 24 or about 24 and 72 hours post infection.

Two highly expressed very late genes have been characterized, the polyhedrin and the p10 gene, and their respective very late promoters have been named polh and p10 promoter. Examples for ACMNPV-derived sequences serving as very late promoters are given as SEQ ID NO: 1 (polh) and SEQ ID NO: 2 (p10). During the very late phase of infection both genes undergo a burst of transcription, leading to accumulation of their respective RNAs and proteins in the cell (Virology 248, 131- 138). Very late promoters are thought to differ from late promoters primarily by the 25 presence of a burst sequence, a sequence downstream of the transcription start site LU103239 that is 90% A and T (Mistretta and Guarino, 2005).

As will be appreciated by the person of ordinary skill, the term “baculovirus late and/or very late promoter’ comprises any promoter that functions as a late and/or functions as a very late promoter for a baculovirus and includes such promoters that comprise nucleic sequences derived from a late, or from a very late, promoter that is present in the genome of a wild type baculovirus. In this context, “derived from” includes nucleic acids sequences that are greater than 80%, such as 85%, 90%, 95%, 99% or 100% identical over about 5, 10, 15, 20, between 20 and 30 or between 30 and about 50 bp.

Amounts of polyhedrin-driven (or another late and/or very late promoter) expression can be easily monitored in cells infected with baculovirus with the gene of interest mutated or deleted or under the control of a weak promoter and compared to cells infected with “wild type” baculovirus at the same M.O.I. by, e.g., immunoblot examination. The person skilled in the art will appreciate that any other method for detecting quantitatively or semi-quantitatively the product of the gene of interest and the late and/or very late gene product (for example polyhedrin or alternatively p10) can be applied. Detection can be at the transcriptional or translational level, detecting

MRNA or protein levels, respectively. Non-limiting examples for detection methods are flow cytometry, microscopy, real-time PCR, immuno- or Western blotting, ELISA and Northern blotting. Further, the polyhedrin or, alternatively, p10 gene or any suitable late and/or very late baculovirus gene, can be replaced by a reporter gene such as a gene encoding chloramphenicol acetyltransferase (CAT), a fluorescent protein like GFP, YFP or their enhanced analogues, a luminescent protein like luciferase or any other protein that is easily detectable. Methods for detecting and quantifying reporter gene expression are well known in the art.

The term “wild type virus” as used herein refers to the phenotype of the typical form of a virus species as it occurs in nature including expression systems derived therefrom. In the case of ACMNPV the wild type virus is encoded by the sequence of

NCBI accession number NC_001623 (Ayres et al. (1994) Virology 202 (2), 586-605) and wild type expression systems are based on this sequence. 26

The abbreviation M.O.1 (or MOI) as used herein refers to the multiplicity of infection and is the ratio of infectious virus particles to infection targets (e.g. cells). For example, when referring to a group of cells inoculated with infectious virus particles, the multiplicity of infection or M.O.l is the ratio defined by the number of infectious virus particles deposited in a well divided by the number of target cells present in that well.

The term “transduce” or “transduction” as used herein refers to the process by which foreign

DNA is introduced into a cell by a viral vector, particularly a mammalian cell by a baculoviral vector.

The invention also provides a composite baculovirus DNA comprising the baculovirus expression system as described herein.

In another aspect, the invention provides a host cell comprising the inventive baculovirus vector system and/or the composite baculovirus DNA of the invention.

Host cells for use in the invention may be any one of a bacterial cell, a yeast cell, a mammalian cell or an insect cell, preferably a bacterial cell or an insect cell. As outlined above, the baculovirus vector system of the invention may comprise several vectors containing one or more elements thereof. Host cells used for propagating individual components, typically one or more transfer vectors comprising, e.g. expression cassettes A and/or B and/or, optionally, C, as well as cells in which one or more of the elements of the baculovirus vector system of the invention are assembles (e.g. fusion of a one or more transfer vectors with a bacmid) are also referred to herein as “intermediate host cells”.

In one preferred embodiment, the host cell of present invention is an insect cell comprising the baculovirus expression system or the composite baculovirus DNA as described herein.

Further, the invention described herein provides a method of delivery of DNA and at least one heterologous gene product, preferably RNA and/or protein as described herein, into a mammalian cell comprising the steps of a) introducing the baculovirus expression system or the composite baculovirus DNA of the invention into insect cells, b) culturing said insect cell allowing viral amplification under conditions where recombinant protein expression enables packaging of heterologous protein and/or 27

RNA into the baculovirus particle, c) harvesting said baculovirus particle d) LU103239 transduction of mammalian cells with said baculovirus particle.

The term “introducing” as used herein refers to any method known to the person skilled in the art to bring the DNA encoding the baculovirus expression systems of the invention into an insect cell. This comprises methods such as transfection, microinjection, transduction and infection. Methods for transfecting DNA into insect cells are known to the person skilled in the art and can be carried out, e.g., using calcium phosphate or dextran, by electroporation, nucleofection or by lipofection.

In the baculovirus expression system of the invention recombinant genes are cloned into the baculovirus genome either through recombination of modified baculovirus

DNA and recombinant gene-containing plasmids in insect or bacteria cells, or through in vitro generation. Using any of these methods, sufficient recombinant protein-encoding viruses or DNA are generated to infect or transfect, respectively, insect cell culture volumes in the order of about a few milliliters.

The term “culturing” as used herein refers to maintaining insect cells in a suitable medium and under conditions allowing the cell to be maintained for the virus to amplify. This also includes up-scaling of virus producing insect cell cultures, comprising serial steps of harvesting virus and reinfecting larger insect cell cultures, or comprising a single step of infecting a cell culture at low M.O.1 and allowing the multiple generations of baculoviral passage to occur in situ by maintaining the culture in log phase cell densities. In certain embodiments culturing comprises the generation of 10, 100, 1000 or even more liters of insect cell culture comprising the inducible baculovirus expression system of the invention.

The insect cell according to the present invention, including an intermediate host insect cell, can be any insect cell supporting baculovirus production. Examples for insect cells supporting baculovirus production are cells derived from Spodoptera frugiperda, Trichoplusia ni, Plutella xylostella, Manduca sexta, and Mamestra brassicae. Preferred insect cells in the context of the invention are the IPLB-SF21AE cell or its clonal isolate, the Sf9 cell.

In yet another aspect the invention provides a kit for a baculovirus expression system, such as in, or for use in, insect and mammalian cells, comprising two or 28 more of elements (1) to (3) of the baculovirus vector system of the invention on a LU103239 transfer vector and a modified baculovirus DNA. The modified baculovirus DNA can be provided in any suitable form, for example as a DNA in a purified form, in a bacterial cell, in a yeast cell, or in an insect cell, in particular said baculovirus DNA comprising expression cassette A. In some embodiments of the invention the kit comprises a transfer vector comprising at least one expression cassette B coding for at least one heterologous gene product such as a heterologous polypeptide. In certain embodiments of the invention the transfer vector may contain instead of an expression cassette encoding a heterologous polypeptide or, in other embodiments in addition thereto, an expression cassette B comprising a nucleotide sequence coding for an RNA into which one or more open reading frame(s) encoding a recombinant protein can be cloned. In some embodiments, the kit further comprises an expression cassette C coding for at least one heterologous gene product such as a heterologous polypeptide, optionally further containing an expression cassette coding for an RNA , which can be located on the transfer vector together with expression cassette(s) B or on the modified baculovirus DNA.

The kits may further comprise instructions for using the components of the kit. Such instructions can be included in the kit in written, electronic, or any other suitable form, describing how to make and employ the recombinant baculovirus vector systems of the present invention.

Typically, baculovirus vector systems provided according to the invention are derived from nuclear polyhedrosis viruses (NPV). While in principle all baculovirus expression systems can be modified to work in the context of the present invention, a preferred inducible baculovirus expression system is based on Autographa californica nuclear polyhedrosis virus (AcMNPV). Examples of other preferred viruses include any of the multiple nucleocapsids per envelope (MNPV) subgenera of the NPV genera of the

Eubaculovirinae subfamily (occluded Baculoviruses) of the Baculoviridae family of insect viruses.

There are a number of commercial systems for expressing recombinant proteins using baculovirus, all based on ACMNPV. They include flashBAC™ (Oxford

Expression Technologies EP1144666), BackPack™ (BD Biosciences Clontech), 29

BacVector® 1000/2000/3000 (Novagen®), BAC-TO-BAC® (Invitrogen™ US LU103239 5,348,886), and BaculoDirect™ (Invitrogen™). All these baculovirus-based insect cell expression systems are based on the strategy of expressing recombinant proteins by placing them under the control of very late baculovirus promoters, namely the polh and/or p10 promoters. Any of these baculovirus expression systems could be conveniently modified to comply with the present invention by incorporating expression cassettes A, B and, optionally, C, into the system as described herein, thereby delivering DNA, RNA and protein into mammalian cells. Accordingly, the baculovirus expression systems of the present invention can be based on any one of the commercially or academically available baculovirus expression systems.

In certain embodiments of the present invention there are provided uses and methods of using, in particular methods of treatment and the use in such treatments, preferably gene therapy, gene editing, gene engineering, cell therapy, immunotherapy, delivery of RNA interference systems, delivery of apoptosis factors, iPS reprogramming, synthetic biology, production of protein complexes, modification of cell signaling, drug discovery, diagnostics, and vaccines, a recombinant nucleic acid, such as one comprising expression cassette(s) A, B and, optionally, C, as defined or otherwise described herein. For example, such a recombinant nucleic acid can comprise at least element(s) (1) and/or (2) and/or (3) of the baculovirus vector system of the invention.

In other embodiments, the inventions also provides nucleic acids prepared for insertion of nucleic acid sequences coding for the modified viral envelope protein of expression cassette A and/or prepared for insertion of nucleic acid sequences coding for the heterologous gene product(s) of expression cassette B and/or, optionally, prepared for insertion of nucleic acid sequences coding for the heterologous gene product(s) of expression cassette C. Typically, such nucleic acids at least comprise the promoter of expression cassette(s) A and/or B and/or, optionally, C, of the baculovirus expression system of the present invention, and (ii) a cloning site, such as one for inserting a nucleic acid coding for said . Suitable cloning/insertion sites for the recombinant nucleic acids of the invention will be readily known to the person of ordinary skill and include (multiple) cloning sites that can be digested by one or more 30 restriction enzyme, and/or recombination-based insertion sites such as those used ~~ LU103239 for the “Gateway” cloning system of Invitrogen of that use Cre-Lox system.

In another aspect, the present invention relates to a vector, such as a bacmid, comprising a recombinant nucleic acid of the invention. In certain embodiments, the vector of the invention is useful for fusion with baculovirus DNA, including with modified baculovirus DNA, and in particular such embodiments the vector is a baculovirus transfer vector. As will be appreciated by the person of ordinary skill, such vector will also comprise other features that assist the maintenance and/or replication of the vector in a cell, such as in a cell described herein.

In yet another aspect, the present invention relates to a composition that includes a one or more vectors as described herein such as one or more vectors of the inventive baculovirus vector system and/or composite baculovirus DNA of the invention and/or baculovirus particles as described herein and/or host cells and/or cell culture according to the invention.

A composition of the present invention includes any mixture of two or more components one of which includes a vector and/or composite baculovirus DNA and/or baculovirus particles and/or host cells and/or cell cultures as defined, claimed or otherwise described herein. In certain embodiments of such aspect, the composition is a two-component mixture including such nucleic acid and at least one other component useful for the construction, and/or practice of the methods using, the baculovirus vector system of the invention. Such other component may include one or more nucleic acids encoding further expression cassettes which may encode homologous or heterologous gene products such as polypeptides and/or

RNA. In other embodiments of this aspect of the invention, the composition may be a complex mixture, such as that of, or otherwise found in, a cell-free transcription/translation system, a cell-extract or an intact cellular environment. In particular such embodiments, the inventive composition that includes a recombinant nucleic acid of the invention is a cell, such as a bacterial, yeast, insect or mammalian cell, for example such a cell comprised in in-vitro or industrial tissue culture or storage.

In a further aspect, the present invention relates to modified baculovirus DNA that comprises expression cassette A of element (1) of the baculovirus vector system of the invention, wherein the expression cassette A contains an open reading frame coding for said modified viral envelope protein. In certain embodiments the modified baculovirus DNA of the invention optionally further comprises expression cassette(s) B such as expression cassette(s) B1 and/or B2, and/or C such as expression cassette(s) B1 and/or B2. 31

As will now be apparent to the person of ordinary skill, the various methods of the present LU103239 invention have particular advantages in the context of large scale (such as industrial) production of recombinant baculovirus. Accordingly, in certain embodiments of the various methods of the invention, the method includes a step of culturing insect cells that comprises conditions under which baculovirus is amplified in said insect cells, under which the number of insect cells increases and/or under which the number of baculovirus particles is increased.

In those methods including a step comprising culture conditions for baculovirus amplification, said conditions comprise between 2 and about 10 rounds of virus amplification, such as comprising about 3, about 4, about 5, about 6 or about 8 rounds of virus amplification.

In certain embodiments of the method claims, the culture conditions are maintained (and/or repeated), or the various steps of the method are repeated until the number of insect cells is between about 108 and about 103, such as between about 10° and 10°? about cells, and/or until the number of baculovirus particles is between 102 and 10", such as between about 10° and about 10°? virus particles. In alternative embodiments, said conditions are maintained until the total volume of culture is from about 0.1 L to about 10,000 L, such as from about 100 to about 1,000 L, and/or are maintained for a period of time from about 1 day to about 3 weeks, such as from about 3 days to about 1 week after the introduction of the baculovirus system of the invention into the insect cell or the provision (and/or start of culture) of the insect cell comprising such a system of the invention.

Other aspects of the present invention relate to a kit, such as a kit of parts, that includes a plurality of components for the construction and/or use of the baculovirus vector system of the present invention. Such plurality of components may be presented, packaged or stored separately. For example, they may be isolated from one another by being held in separate containers. Accordingly, one embodiment of such a kit of the invention comprises at least two components that include (preferably separately): (i) the recombinant nucleic acid, vector, composition or the modified composite baculovirus DNA of the invention; and (ii) at least one other component for the construction and/or use of an inducible baculovirus expression system. Such components, although held separately, may be boxed or otherwise associated together to aid storage and/or transport, and such association may include additional components. In particular embodiments of such kits of the invention, at least one of the second (or additional) components may comprise: (i) the expression cassette(s) B (such as one or more expression cassettes B1 encoding at least one heterologous polypeptide and/or one or more expression cassettes B2 coding for a (untranslated) RNA) and/or C (such as one or more expression cassettes C1 encoding at least one heterologous polypeptide and/or one or more expression cassettes C2 coding for a (untranslated) RNA) of the baculovirus 32 vector system of the invention, and/or a vector and/or a cell that comprises said expression LU103239 cassette(s); (ii) an insect cell, preferably one selected from the selected from the group consisting of insect cells derived from Spodoptera frugiperda, Trichoplusia ni, Plutella xylostella, Manduca sexta, and Mamestra brassicae, such as an insect cell that is a IPLB-

SF21AE cell or its clonal isolate Sf9; (iii) an inducer molecule that modulates the reversible interaction of a controllable transcriptional modulator protein with a transcriptional modulator response element; and, optionally (iv) instructions describing how to construct and/or use the inducible baculovirus expression system of the present invention, and/or to practice any of the various methods of the present invention.

The invention is further directed to the following aspects (A) A further aspect of the invention is an AcMNPV baculovirus vector system for mammalian cells, comprising an ACMNPV baculovirus genome and: a) at least one heterologous expression cassette A which is incorporated in the baculovirus genome, containing a promoter and an open reading frame coding for at least one modified viral envelope protein which is expressed in insect cells and incorporated into the baculovirus particle upon virus production in insect cells; and b) at least one further heterologous expression cassette D which is incorporated in the baculovirus genome, and coding for at least one heterologous gene product such as one or more polypeptide(s) which is/are expressed in mammalian cells (in which case the respective expression cassette(s) comprise(s) a promoter and an open reading frame coding for the heterologous polypeptide(s) and may be denoted as “expression cassette(s) D1), and/or one or more (untranslated) RNAs (in which case the respective expression cassette(s) contain(s) a promoter which transcribes the heterologous RNAs in mammalian cells and may be denoted as expression cassette(s) D2); and c) deletion of the p94 gene (SEQ ID NO: 12), v-chiA (SEQ ID NO: 19) and v-cath (SEQ ID NO: 16) genes from the AcMNPV baculovirus genome; and d) wherein the baculovirus genome contains an attTn7 Transgene Insertion Site, preferably comprising, essentially consisting of, or consisting of attTn7L (SEQ ID

NO: 17) and attTn7R (SEQ ID NO:18), within 4000 bps of homologous repeated (hr) sequence hr2, hr3, hr4b, or the odv-e56 (pif-5) locus (SEQ ID NO: 15). 33

According to a preferred embodiment of aspect (A) the total combined size of LU103239 heterologous DNA cargo comprising the expression cassettes A and D (such as comprising expression cassettes D1 and/or D2 is >10°000 bps, preferably >15000 bps, more preferably >20°000 bps. (B) The invention is also directed to an autographa califomica multicapsid nucleopolyhedrovirus (AcMNPV) bacmid shuttle vector (system), comprising: a) the AcMNPV baculovirus genome which includes the elements required for said baculovirus vector propagation in insect cells, but is lacking the p94 (SEQ ID NO: 12) and v-cath (SEQ ID NO: 16) genes; and b) an attTn7 Transgene Insertion Site, preferably comprising, essentially consisting of, or consisting of attTn7L (SEQ ID NO: 17) and attTn7R (SEQ ID NO:18), imbedded in said ACMNPV genome, where said insertion site is located within 4000 bps of homologous repeated (hr) regions hr2, hr3, hr4b or the odv-e56 (pif- 5) locus (SEQ ID NO: 15); and c) a bacterial replicon which is located more than 5000 base pairs distant in said

AcMNPV genome from the attTn7 Transgene Insertion Site of b) where said bacterial replicon is capable of driving the replication of said ACMNPV bacmid shuttle vector in bacteria; and d) a bacterial genetic marker which is located more than 5000 base pairs distant in the ACMNPV genome from the attTn7 Transgene Insertion Site of b). (C) Another aspect of the invention relates to an ACMNPV baculovirus vector system for insect cells, comprising: a) atleast one heterologous expression cassette A containing a promoter and an open reading frame coding for at least one heterologous polypeptide(s) which is expressed in insect cells from a loci within 4000 bps of the baculovirus ORF 1629; and b) the AcMNPV baculovirus genome comprises a deletion of the p94 gene (SEQ ID

NO: 12) and/or the v-chiA (SEQ ID NO: 19) and v-cath genes (SEQ ID NO: 16) ; and c) atleast one additional heterologous expression cassette B, containing a promoter and an open reading frame coding for at least one heterologous polypeptide(s) which is expressed in insect cells from a loci within 4000 bps of 34 the deleted p94 gene and/or the v-chiA and v-cath genes of b) or which is LU103239 expressed in insect cells from a loci within 4000 bps of homologous repeated (hr) sequence hr2, hr3, hr4b or the odv-e56 (pif-5) gene (SEQ ID NO: 15).

Preferably, the baculovirus vector systems according to above-defined aspects (B) and (C) are provided for use in biopharmaceutical manufacturing, or for production of rAAV vectors. (D) An AcMNPYV baculovirus vector system for mammalian cells, comprising an

AcMNPV baculovirus genome and: a) at least one heterologous expression cassette A which is incorporated in the baculovirus genome, containing a promoter and an open reading frame coding for at least one modified viral envelope protein which is expressed in insect cells and incorporated into the baculovirus particle upon virus production in insect cells; and b) at least one heterologous expression cassette B which is incorporated in the baculovirus genome, containing a promoter and an open reading frame coding for at least one immune complement system inactivating factor ; and c) at least one further heterologous expression cassette C incorporated in the baculovirus genome and encoding at least one heterologous gene product (such as one or more expression cassettes incorporated in the baculovirus genome and coding for heterologous polypeptide(s) which is expressed in mammalian cells (which may be denoted as expression cassette C1), and/or at least one expression cassette incorporated in the baculovirus genome and containing a promoter which transcribes a heterologous (untranslated) RNA in mammalian cells (which may be denoted as expression cassette C2); and d) deletion of the v-chiA (SEQ ID NO: 19) and v-cath (SEQ ID NO: 16) genes from the ACMNPV baculovirus genome; wherein upon transduction of mammalian cells with said baculovirus particle, the heterologous polypeptide(s) and/or RNA(s) produced by c) modulates at least one key physiological process of said mammalian cells. And wherein the total combined size of heterologous DNA cargo comprising the expression cassettes A and C (such as C1 and/or C2) is >10°000 bps, preferably >15000 bps, more preferably >20’000 bps.

In preferred embodiments of aspects (A) to (D) the key physiological process of said mammalian cells modulated by the product(s) of expression cassette C (such as C1 and/or C2) include genome modifications, intracellular organization, metabolism, responsiveness, signaling, movement, reproduction, or death.

Preferably, metabolism includes intracellular trafficking, RNA transcription, protein translation, or cellular energy levels.

Preferably, genome modifications organization include permanent sequence alterations to host cell chromosomal DNA.

Preferably, the modified viral envelope protein, is selected from the group consisting of VSV-G, HIV gp120, baboon envelope protein, and modified versions of baculovirus envelope protein GP64.

Preferably the modified viral envelope protein of aspects (A) to (D) is VSV-G of SEQ

ID NO: 3 or a transcriptionally functional homolog thereof, more preferred Preferably the transcriptionally functional homolog of VSV-G is at least 70 % identical on the amino acid level to VSV-G of SEQ ID NO: 3, more preferably at least 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 % identical on the amino acid level to VSV-G of SEQ ID

NO: 3.

Preferably, the immune complement system inhibitor is selected from the group consisting of decay-accelerating factor (DAF), factor H (FH)-like protein-1 (FHL-1),

C4b-binding protein (C4BP), and membrane cofactor protein (MCP).

Preferably, the immune complement system inhibitors are displayed on the baculovirus surface via fusions to baculovirus envelope protein GP64 (SEQ ID NO: 13), or the membrane anchor of the vesicular stomatitis virus-G (VSV-G) protein (SEQ ID NO: 14).

Preferably, the immune complement system inhibitor fusion to baculovirus envelope protein GP64 is the DAF-GP64 fusion protein according to SEQ ID NO: 15). 36

Preferably, the immune complement system inhibitor fusion to baculovirus envelope protein GP64 is the DAF-VSV-G membrane anchor fusion protein according to SEQ

ID NO: 16.

Preferably, the baculovirus vector system of any one of aspects (A) to (D), is provided for use in gene therapy, gene editing, gene engineering, cell therapy, immunotherapy, delivery of RNA interference systems, delivery of apoptosis factors, iPS reprogramming, synthetic biology, production of protein complexes, modification of cell signaling, drug discovery, diagnostics, and vaccines.

All references cited are incorporated by reference in their entirety to the extent that they are not inconsistent with the description herein.

The present invention is further illustrated by the following non-limiting Examples:

EXAMPLES

Example 1

Example 1 illustrates the production in SF21 insect cells of a baculovirus particle pseudotyped with the VSV-G envelope glycoprotein, and loaded with mCherry protein also produced in insect cells. The resulting particle thereafter delivers into mammalian cells mCherry protein together with a heterologous DNA expression cassette coding for eGFP following transduction of said mammalian cells.

As shown in Fig. 1 (top), the VSV-G envelope glycoprotein (SEQ ID NO: 3.) is the product of expression cassette A). The product of expression cassette B) is the mCherry protein (SEG

ID NO: 4.). Expression cassette C) codes for the enhanced green fluorescent protein, eGFP (SEQ ID NO: 11), under control of the mammalian hPGK promoter (SEQ ID NO: 5.).

Expression cassette A) coding for the VSV-G envelope glycoprotein is under control of the p10 promoter (SEQ ID NO: 2.), which causes it to be produced in said insect cells, where it becomes associated with the envelope of the baculovirus particle (see Barsoum et al. (1997), and Mangor et al. (2001)) Fig. 1 (middle). Similarly, mCherry (SEQ ID NO: 4.) is simultaneously produced in said insect cells under the p10 promoter (SEQ ID NO: 2.) and thereby loaded into the baculovirus particle in insect cells (Fig. 1, middle). Expression 37 cassette C) coding for the fluorescent protein eGFP is integrated into the DNA viral genome LU103239 of the baculovirus particle Fig. 1 (middle).

Upon harvesting of said produced baculovirus particles from insect cells, the viruses are then used to transduce mammalian CHO cells Fig. 1 (bottom). Upon transduction of the CHO cells by said baculovirus particles, mCherry protein is delivered into the CHO cells, while

Expression cassette C) is transcribed and translated by the CHO cells to produce eGFP inside the CHO cells.

As shown in Fig. 2, transfer vector pEXAMPLE1.1 containing expression cassette C) coding for eGFP under the hPGK promoter and expression cassette B) coding for mCherry under the p10 promoter was constructed starting with plasmid pFL from (Fitzgerald et al., 2006). To construct pEXAMPLE1.1 expression cassette C) coding for eGFP under the hPGK promoter was constructed by gene synthesis and cloned into the EcoRI site of plasmid pFL. Next, the coding sequence of mCherry was constructed by gene synthesis and cloned into the Xhol site of plasmid pFL.

The strategy for creation of composite baculovirus containing expression cassettes A), B), and C) is described schematically in Fig. 3. Expression cassette A) coding for the VSV-G envelope glycoprotein under control of the p10 promoter was integrated into the

DH10MultiBac genome (Berger et al, 2004) as described (Monsouri et al., 2016) to create baculovirus DNA DH10EMBacVSV (Fig. 3, left). Composite baculovirus DNA containing expression cassettes A), B), and C) is created by fusing transfer vector pEXAMPLE1.1 with

DH10EMBacVSV cells. This is carrie42d out by transforming pEXAMPLE1.1 into

DH10EMBacVSV E. coli cells harboring the bacmid DH10EMBacVSV and the helper plasmid pMON712417. Tn7 mediated recombination in the DH10EMBacVSV cells then mediates the fusion of DH10EMBacVSV with pEXAMPLE1.1 as described in (Berger et al., 2004).

Composite bacmid DNA is isolated from DH10EMBacVSV cells following Tn7 transposition and transfected into SF21 insect cells according to (Fitzgerald et al., 2006). Initial transfection virus is amplified for 2 passages as described (Fitzgerald et al., 2006) and used for subsequent transduction experiments of CHO cells.

Transduction experiments of CHO cells are carried out as follows. CHO cells cultured in CD

OptiCHO™ medium (ThermoFischer) were seeded in 24 well plates (0.5 ml per well) and these adherent cells incubated at 37°C, 5% CO2 for 24h. The cells were then transduced with the different baculovirus viruses at 5:1 multiplicity of infection (MOI), followed by 3h incubation at 25°C on a plate rocker at 30 motions per minute. Following the 3h incubation, 38 the transduction suspension was quantitatively removed and replaced by fresh CHO culture LU1032539 media. The cells were then incubated at 37°C, 5% CO2 for a further 24h. Analysis of cells via

Fluorescence-Activated Cell Sorting (FACS) was as described in (Kim et al, 2012).

The cluster corresponding to CHO cells was identified (Figure 4A, Left), and only the events falling in this region were kept for analysis. The threshold for fluorescence (Figure 4A, right) was determined by setting the percentage of fluorescent cells in this sample identical to a non-transduced sample run as a control close to zero (typically around 0.1 to 1.0% of cells where this small percentage appears to be due to visible autofluorescence of dying cells or debris). In this example (Figure 4A, right) 89.58 % of the cells were fluorescent green.

Simultaneous delivery by a baculovirus particle of mCherry protein into CHO cells with a

DNA expression cassette encoding eGFP was demonstrated using the FACS method described in Figure 4A. CHO cells cultured as described in Figure 4A were transduced with the virus described in Figure 3 at different MOIs and analyzed by FACS. FACS was used to determine the % of cells containing eGFP (Figure 4B left) or mCherry (Figure 4B, right), as a function of increasing MOI. As can be seen in Figure 4B, >80% delivery/transduction of both EGFP and mCherry can be achieved with MOIs of 5 and above.

Example 2

The inducible caspase-9 suicide (iCasp9) gene system acts as a “safety switch” to limit on- target, off-tumor toxicities of Chimeric Antigen Receptor (CAR) T cell therapies (Gargett and

Brown, 2014). Co-delivery of safety switches together with CARs is an important industrial application in the field of oncology. Example 2 illustrates the production in SF21 insect cells of a baculovirus particle pseudotyped with the VSV-G envelope glycoprotein, and loaded with iCasp9 protein, also produced in insect cells. The resulting particle thereafter delivers into mammalian Jurkat cells iCasp9 protein together with a heterologous DNA expression cassette encoding a CAR.

As shown in Fig. 5 (top), expression cassette A) coding for the VSV-G envelope glycoprotein is under control of the p10 promoter (SEQ ID NO: 2.), which causes it to be produced in said insect cells, where it becomes associated with the envelope of the baculovirus particle. The product of expression cassette B) named in the figure as “iCasp9” is the inducible caspase 9- based suicide protein from (doi: 10.1182/blood-2004-11-4564). iCasp9 is simultaneously produced in said insect cells under the p10 promoter (SEQ ID NO: 2.) and thereby loaded into the baculovirus particle in insect cells (Fig. 5, middle). Expression cassette C) codes for the anti-CD19 CAR protein, from (DOI: 10.1038/sj.leu. 2403302), under control of the 39 mammalian hPGK promoter (SEQ ID NO: 5.) and is integrated into the DNA viral genome of LU103239 the baculovirus particle Fig. 5 (middle).

Upon harvesting of said produced baculovirus particles from insect cells, the viruses are then used to transduce mammalian Jurkat cells Fig. 5 (bottom). Upon transduction of the Jurkat cells by said baculovirus particles, iCasp9 protein is delivered into the Jurkat cells, while

Expression cassette C) is transcribed and translated by the Jurkat cells to produce the CAR protein inside the Jurkat cells.

As shown in Fig. 6, transfer vector pEXAMPLE2.1 containing expression cassette C) coding for the CAR under the hPGK promoter and expression cassette B) coding for iCasp9 under the p10 promoter was constructed starting with plasmid pFL (Fitzgerald et al., 2006). To construct pEXAMPLE2.1 expression cassette C) coding for the CAR under the hPGK promoter was constructed by gene synthesis and cloned into the EcoRl site of plasmid pFL.

Next, the coding sequence of iCasp9 was constructed by gene synthesis and cloned into the

Xhol site of plasmid pFL.

The strategy for creation of composite baculovirus containing expression cassettes A), B), and C) is described schematically in Fig. 7. Expression cassette A) coding for the VSV-G envelope glycoprotein under control of the p10 promoter was integrated into the

DH10MultiBac genome (Berger et al, 2004) as described (Monsouri et al., 2016) to create baculovirus DNA DH10EMBacVSV (Fig. 7, left). Composite baculovirus DNA containing expression cassettes A), B), and C) is created by fusing transfer vector pEXAMPLE2.1 with

DH10EMBacVSV cells. This is carried out by transforming pEXAMPLE2.1 into

DH10EMBacVSV E. coli cells harboring the bacmid DH10EMBacVSV and the helper plasmid pMON712417. Tn7 mediated recombination in the DH10EMBacVSV cells then mediates the fusion of DH10EMBacVSV with pEXAMPLE2.1 as described in (Berger et al. 2004).

Composite bacmid DNA is isolated from DH10EMBacVSV cells following Tn7 transposition and transfected into SF21 insect cells according (Fitzgerald et al., 2006). Initial transfection virus is amplified for 2 passages as described (Fitzgerald et al, 2006) and used for subsequent transduction experiments of Jurkat cells.

Transduction experiments of Jurkat cells are carried out as follows: the Jurkat E6.1 T cell line were thawed following the protocol given by the supplier and resuspended in medium supplemented with Interleukin-2 (RPMI, 10% FCS, 1% P/S, 100 IU/ml human recombinant

Interleukin-2 (hr-IL-2)) at a concentration of 10e6 cells/ml. hr-IL-2 is added to the medium to support the growth of T cells and acts as a survival signal and helps maintaining primary T lymphocytes in vitro. The cells were then seeded in 24 well plates (0.5 ml per well) and LU103239 incubated at 37°C, 5% CO2 for 24h. The cells were then transduced with the virus described above.

Quantification of the percent of Jurkat cells expressing the CAR was carried out using FACS and the REAfinity™ Recombinant Technology (Miltenyi Biotec) according to the manufacturer’s instructions. Figure 8, left, shows the % of cells expressing the CAR as a function of increasing MOI.

Cell death through iCasp9 can be triggered by the small molecule chemical AP1903, where addition of AP1903 Kills cells in less than an hour. The protocol utilized for triggering cell death via AP1903, and measuring dead Jurkat cells via FACS is described in (Zhou et al, 2015) Figure 8, right, shows the % of Jurkat cells that were killed as a function of increasing

MOI.

As can be seen in Figure 8, > 80% delivery/transduction of both iCasp9 and CAR can be achieved with MOIs of 5 and above.

Example 3

Co-delivery or co-production of multiple components in one cell is a necessary prerequisite for gene editing applications such as CRISPR. Example 3 illustrates the production in SF21 insect cells of a baculovirus particle pseudotyped with the VSV-G envelope glycoprotein, a genome editing protein, Cas9-GFP, and its guide RNAs, all of which are co-produced in insect cells. The resulting particle thereafter delivers Cas9-GFP protein, guide RNAs, and an expression cassette coding for the interleukin IL-2, fused with the fluorescent protein RFP (eRFP-IL2).

As shown in Fig. 9 (top), expression cassette A) coding for the VSV-G envelope glycoprotein is under control of the p10 promoter (SEQ ID NO: 2.), which causes it to be produced in said insect cells, where it becomes associated with the envelope of the baculovirus particle. The product of expression cassette B1) named in the figure as “Cas9-GFP” is a fusion protein of

Cas9 (SEQ ID NO: 6.) and eGFP (SEQ ID NO: 11.). The product of expression cassette B2) named in the figure as “Guide RNAs” are HMGA1-gRNA1 from (Monsouri et al., 2016) which are simultaneously produced in said insect cells under the p10 promoter (SEQ ID NO: 2.) and thereby loaded into the baculovirus particle in insect cells (Fig. 9, middle). Expression cassette C) codes for the eRFP-IL2 coding sequence under the control of the mammalian 41 hPGK promoter (SEQ ID NO: 5.) and is integrated into the DNA viral genome of the LU103239 baculovirus particle Fig. 9 (middle).

Upon harvesting of said produced baculovirus particles from insect cells, the viruses are then used to transduce mammalian CHO cells Fig. 9 (bottom). Upon transduction of the CHO cells by said baculovirus particles, Cas9-GFP protein and the guide RNA HMGA1-gRNA1 are both delivered into the CHO cells, while Expression cassette C) is transcribed and translated by the CHO cells to produce the eRFP-IL2 protein inside the CHO cells.

As shown in Fig. 10, transfer vector pEXAMPLE3.1 containing expression cassette C) coding for the eRFP-IL2 under the hPGK promoter and expression cassette B1) coding for

Cas9-GFP under the p10 promoter, and B2) guide RNAs are under the p10 promoter are indicate was constructed starting with plasmid pFL from (Fitzgerald et al., 2006). To construct pEXAMPLES.1 expression cassette C) coding for eRFP-IL2 under the hPGK promoter was constructed by gene synthesis and cloned into the EcoRI site of plasmid pFL. Next, the coding sequence of B1) Cas9-GFP was constructed by gene synthesis and cloned into the

Xhol site of plasmid pFL. Finally, B2) guide RNAs under the control of the p10 promoter was constructed by gene synthesis and cloned into the Pmel site of pFL.

The strategy for creation of composite baculovirus containing expression cassettes A), B1),

B2) and C) is described schematically in Fig. 11. Expression cassette A) coding for the VSV-

G envelope glycoprotein under control of the p10 promoter was integrated into the

DH10MultiBac genome (Berger et al. 2004) as described (Monsouri et al, 2016) to create baculovirus DNA DH10EMBacVSV (Fig. 11, left). Composite baculovirus DNA containing expression cassettes A), B1), B2) and C) is created by fusing transfer vector pEXAMPLES.1 with DH10EMBacVSV cells. This is carried out by transforming pEXAMPLE3.1 into

DH10EMBacVSV E. coli cells harboring the bacmid DH10EMBacVSV and the helper plasmid pMON712417. Tn7 mediated recombination in the DH10EMBacVSV cells then mediates the fusion of DH10EMBacVSV with pEXAMPLE2.1 as described in (Berger et al. 2004).

Composite bacmid DNA is isolated from DH10EMBacVSV cells following Tn7 transposition and transfected into SF21 insect cells according to (Fitzgerald et al., 2006). Initial transfection virus is amplified for 2 passages as described (Fitzgerald et al., 2006) and used for subsequent transduction experiments of CHO cells. 42

Cell culture and transduction experiments of CHO cells were carried out as described in LU103239

Example 1. The cells were transduced with increasing MOIs of virus described above in

Examples 1 and 2.

Quantification of the percent of CHO cells expressing the fluorescent fusion proteins Cas9-

GFP and eRFP-IL2 was carried out using FACS as described in Example 1. Figure 12, top, shows the % of cells expressing the Cas9-GFP as a function of increasing MOI. Figure 12, center, shows the % of cells expressing the eRFP-IL2 as a function of increasing MOI.

Figure 12, bottom, shows the % of cells containing guide RNAs as measured by fluorescent in-situ hybridization to detect the guide RNAs by FACS (Young et al., 2020).

As can be seen in Figure 12, > 80% co-delivery of protein, DNA, and RNA can be achieved with MOIs of 5 and above.

Nucleotide and Amino Acid Sequences

Unless otherwise stated, all nucleotide sequences disclosed in the present application are indicated from the 5’ to the 3’ end, and all amino acid sequences disclosed in the present application are indicated from the N-terminus to the C-terminus.

SEQ ID NO: 1: Polh promoter DNA sequence

ATCATGGAGA TAATTAAAAT GATAACCATC TCGCAAATAA ATAAGTATTT TACTGTTTTC

GTAACAGTTT TGTAATAAAA AAACCTATAA ATATTCCGGA TTATTCATAC CGTCCCACCA

TCGGGCGCG

SEQ ID NO: 2: P10 promoter DNA sequence

TATACGGACC TTTAATTCAC CCAACACAAT ATATTATAGT TAAATAAGAA TTATTATCAA

ATCATTTGTA TATTAATTAA AATACTATAC TGTAAATTAC ATTTTATTTA CAATCACTCG

ACG

SEQ ID NO: 3: VSV-G amino acid sequence

MKCLLYLAFLFIGVNCKFT IVFPHNQK&NWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAI

QADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVT

DAEAVIVQVTPHHVLVDEY TGEWVDSQFINGKCSNY ICPTVHNSTTWHSDYKVKGLCDSNLISMDITF

FSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAARARFPECPEG

SSISAPSQTSVDVSLIQDVERILDY SLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLK

YFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGML

43

DSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLII LU103239

GLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

SEQ ID NO: 4: mCherry amino acid sequence

MVLAVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDI

LSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQODGEFIYKVKLRGTN

FPSDGPVMQKKTMGWEASSERMY PEDGALKGE IKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVN

IKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

SEQ ID NO: 5: human phosphoglycerate kinase 1 promoter (hPGK promoter) DNA sequence

GGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTG

GTTCCGGGARACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACC

CGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGG

TTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAG

ACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCA

GCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTG

GGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCARGCCTCCGGAGCGCACGTCGGCAGTCGGCT

CCCTCGTTGACCGAATCACCGACCTCTCTCCCCAG

SEQ ID NO: 6: Cas-9 (Streptococcus pyogenes serotype M1) amino acid sequence

MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE

ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG

NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD

VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN

LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNISDAI

LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA

GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSI PHQIHLGELH

AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE

VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL

SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI

IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ LKRRRYTGWG

RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL

HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER

MKRIEEGIKE LGSQILKEHP VENTQLONEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDH

IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNAK LITQRKFDNL

TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS

KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK

MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF

44

ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA LU103239

YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK

YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE

QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA

PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD

SEQ ID NO: 7: dCas-9 (Staphylococcus aureus) amino acid sequence

MDKKYSIGLA IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE

ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG

NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD

VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN

LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNISDAI

LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA

GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSI PHQIHLGELH

AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE

VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL

SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI

IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ LKRRRYTGWG

RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL

HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER

MKRIEEGIKE LGSQILKEHP VENTQLQONEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDA

IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNAK LITORKFDNL

TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS

KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK

MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF

ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA

YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK

YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE

QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA

PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD

SEQ ID NO: 8: PE1 amino acid sequence (cf. Anzalone, et al, 2019)

DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR

RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYH

LRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASG

VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDD

DLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRY DEHHQDLTLLKALVRQ

QLPEKYKEIFFDQSKNGYAGY IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS

IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN LU103239

FEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFISGEQK

KAIVDLLFKTNRKVTVKQOLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENE

DILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY TGWGRLSRKLINGIRDKQSGKT ILD

FLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK

VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLONEKLY LY YLQ

NGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ

LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREV

KVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM

IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM

PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKY GGFDSPTVAY SVLVVAKVEKGKSKKL

KSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNE

LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSA

YNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID

LSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSD

FPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLIDQGILVPCQSPWNT

PLLPVKKPGTNDYRPVQODLREVNKRVEDIHPTVPNPYNLLSGLPPSHQOWYTVLDLKDAFFCLRLHPTS

QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATS

ELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKY LGY LLKEGQRWLTEARKETVMGQPTPKTPRQL

REFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFEL

FVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA

PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG

TRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELTALTQALKM

AEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQK

GHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV

SEQ ID NO: 9: PE2 amino acid sequence (cf. Anzalone, et al., 2019)

DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR

RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYH

LRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQOTYNQLFEENPINASG

VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDD

DLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRY DEHHQDLTLLKALVRQ

QLPEKYKEIFFDQSKNGYAGY IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS

IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEET ITPWN

FEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQK

KAIVDLLFKTNRKVTVKQOLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENE

DILEDIVLTITLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILD

FLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK

46

VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLONEKLYLYYLQ LU103239

NGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ

LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREV

KVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM

IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGE IVWDKGRDFATVRKVLSM

PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKY GGFDSPTVAY SVLVVAKVEKGKSKKL

KSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNE

LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSA

YNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID

LSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSD

FPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLIDQGILVPCQSPWNT

PLLPVKKPGTNDYRPVQODLREVNKRVEDIHPTVPNPYNLLSGLPPSHQOWYTVLDLKDAFFCLRLHPTS

QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATS

ELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKY LGY LLKEGQRWLTEARKETVMGQPTPKTPRQL

REFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFEL

FVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA

PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG

TRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELTALTQALKM

AEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHOK

GHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEPKKKRKV

SEQ ID NO: 10: p53 protein amino acid sequence

MEEPQSDPSV EPPLSQETFS DLWKLLPENN VLSPLPSQAM DDLMLSPDDI

EQWFTEDPGP DEAPRMPEAA PPVAPAPAAP TPAAPAPAPS WPLSSSVPSQ

KTYQGSYGFR LGFLHSGTAK SVTCTYSPAL NKMFCQLAKT CPVQLWVDST

PPPGTRVRAM AIYKQSQHMT EVVRRCPHHE RCSDSDGLAP PQHLIRVEGN

LRVEYLDDRN TFRHSVVVPY EPPEVGSDCT TIHYNYMCNS SCMGGMNRRP

ILTIITLEDS SGNLLGRNSF EVRVCACPGR DRRTEEENLR KKGEPHHELP

PGSTKRALPN NTSSSPQPKK KPLDGEYFTL QIRGRERFEM FRELNEALEL

KDAQAGKEPG GSRAHSSHLK SKKGQSTSRH KKLMFKTEGP DSD

SEQ ID NO: 11: enhanced green fluorescent protein (eGFP) amino acid sequence

MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGV

QCFSRYPDHMKQOHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNI

LGHKLEYNYNSHNVY IMADKQKNGIKVNFKIRHNIEDGSVQLADHYQONTPIGDGPVLLPDNHYLSTQ

SALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

47

SEQ ID NO: 12: Autographa californica nucleopolyhedrovirus p94 protein: amino acid LU103239 sequence

MINYYVYATDDSLSTNSDYYFNKNALQOTLEQFQNETENISSCDKVLYLHWSAYCRQKEIGDVKSRYLR

RNGEGTDTRPSEFIKWIHKNINLLDGKLKLLYMVTDGQISKNEANVCKNLLNEKPFSFERIVFYAINN

NTEQIDLSVASAFVNNSDCKIYRNDEMVEWVNLTKEFNYDIITTENFISKKDELLSFVRFKFINSMPT

DANVLNEVDKLKRLRQORLFSEIKQOTNNSSMNFDQIKNKNEFVNTFKSTEFYKTLYNADVLINFDKIIDS

TISTAINYLHNRNKSYAFDVMKNLHYQNKLASVNAETDDVVANDDDEAYDY SNVENIRFPDCILANDS

GVPAILLTHYNLFETIQGSLTKFKSRLEFPLLWSQNKEIKNSIEYCYNLESLKQLIQHGTRLSPRSRR

PFTGAIVPNEQFDEYNDYVLACTY FDAKKVAFNAGLMYYLLYKHINDAEYIDDNVKDYFKRYVIYRIN

NTECMIGFSNLAMEPLIKVKLPTALWYVSEISTLLFKHDNQHFGKEKLRQFAHFAEDMLQILQWCDYT

DVNVEAVKKRAYCLKRINMFKRMSVLDAVEWIANRAFECKDKFI INKLTNADALQDLKFLKVNHNGVV

DEHVLNDTSINAERYLYFYHIIEDFDKY ISVVDNTMRPAFVLEEGKTFYDSLLKQLQSVHENGQEITF

EKCSRLDFNRILSLHKLYIECVKSLNKYPTLEEYQNYVYNQKHVKENRIATIFPENILONLAAVHNEYA

NKIVNLPVEEFIVRANNTVNRITRIQNERVGSPLQAEEIDKLIKLSEQRVNICRK

SEQ ID NO: 13: baculovirus envelope protein GP64 amino acid sequence

GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATG

GAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGECTGACCGCCCAACGACCCCCGCCCATT

GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGG

ACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATT

GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTAC

TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGETTTTGGCAGTACATCAATG

GGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTG

TTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGG

CGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT

SEQ ID NO: 14: membrane anchor of the vesicular stomatitis virus-G (VSV-G) protein: amino acid sequence

TASFFFIIGLIIGLFLVLRVGIHLC

SEQ ID NO: 15: Autographa californica nucleopolyhedrovirus odv-e56 (pif-5) protein: amino acid sequence

MSFFSNLRAVNKLYPNQASFITDNTRLLTSTPAGFTNVLNAPSVRNIGNNRFQPGYQLSNNQFVSTSD

INRITRNNDVPNIRGVFQGISDPQINSLSQLRRVDNVPDFNYHTKQOTRSNAVKONFPETNVRTPEGVQ

NALQQONPRLHSYMQSLKVGGTGILLATGGYFLFSAATLVQDI INAINNTGGSYYVQGKDAGETAEACL

LLORTCRODPNINQSDVTICPFDPLLPNNPPELTNMCQGFNYEVEKTVCRGSDPSADPDSPQYVDISD

LPAGQTLMCIEPYSFGDLVGDLGLDWLLGDEGLVGKSSNVSDSVSGKLMPIILLIGAVLFLGLIFYFI

YRYMMKGGGGGGVGAATSPTPIVISMONPTPTTAPR

48

SEQ ID NO: 16: Autographa californica nucleopolyhedrovirus v-cath protein: amino acid sequence

MNKILFYLFV YGVVNSAAYD LLKAPNYFEE FVHRFNKDYG SEVEKLRRFK IFQHNLNEIT

NKNQNDSAKY EINKFSDLSK DETIAKYTGL SLPIQTQNFC KVIVLDQPPG KGPLEFDWRR

LNKVTSVKNQ GMCGACWAFA TLASLESQFA IKHNQLINLS EQQMIDCDFV DAGCNGGLLH

TAFEAIIKMG GVQLESDYPY EADNNNCRMN SNKFLVQVKD CYRYITVYEE KLKDLLRLVG

PIPMAIDAAD IVNYKQGIIK YCFNSGLNHA VLLVGYGVEN NIPYWTFKNT WGTDWGEDGF

FRVQOQNINAC GMRNELASTA VIY

SEQ ID NO: 17 and SEQ ID NO: 18: the attTn7 transgene insertion site DNA sequence comprises a left and right end known as “attTn7L” and “attTn7R”, respectively. It is understood that the combination of SEQ ID NO:17 followed at its 3’ end directly by SEQ ID NO: 18 results inthe complete attTn7 transgene DNA sequence: attTn7L DNA sequence (SEQ ID NO: 17):

CTCTTTTTCGAATGCTTCCATCAGTTTAGATTTATCCATTCATTATGCAATGCTCTCTTCC

— attTn7R DNA sequence (SEQ ID NO: 18):

TGGGAGCCTGTGTAGGCTGGAAGCTGGCGCTGAACTGAGGCACGGGTCAGTATCCGTCGTGT

SEQ ID NO: 19: Autographa californica nucleopolyhedrovirus v-chiA protein: amino acid sequence

MLYKLLNVLWLVAVSNATIPGTPVIDWADRNYALVEINYEATAYENLIKPKEQVDVQVSWNVWNGDIGD

TAYVLFDEQQVWKGDAESKRATIKVLVSGQFNMRVKLCNEDGCSVSDPVLVKVADTDGGHLAPLEYTW

LENNKPGRREDKIVAAY FVEWGVYGRNFPVDKVPLPNLSHLLYGFIPICGGDGINDALKTIPGSFESL

QRSCKGREDFKVAIHDPWAAVQKPQKGVSAWNEPYKGNFGQLMAAKLANPHLKILPSIGGWTLSDPFY

FMHDVEKRNVFVDSVKEFLQOVWKFFDGVDIDWEFPGGKGANPSLGDADGDAKTYILLLEELRAMLDDL

EAQTGRVYELTSAISAGYDKIAVVNYAEAQKSIGKI FLMSYDFKGAWSNTDLGYQTTVYAPSWNSEEL

YTTHYAVDALLKQGVDPNKIIVGVAMYGRGWTGVTNYTNDNY FSGTGNGPGSGTWEDGVVDYRQIQKD

LNNYVYTFDSAAQASYVFDKSKGDLISFDSVDSVLGKVKYVDRNKLGGLFAWE IDADNGDLLNAINAQ

FKPKDEL

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Claims (80)

Our Ref.: G 0180 LU Date: 6 February 2024 LU103239 Applicant: GBiotech Särl Claims

1. A baculovirus vector system comprising one or more recombinant vectors comprising the following elements: (1) a baculovirus genome sufficient for producing baculovirus particles upon transfection in susceptible insect cells; (2) at least one heterologous expression cassette À containing a promoter and an open reading frame coding for at least one modified viral envelope protein, the expression cassette enabling the expression of the at least one modified viral envelope protein in insect cells and incorporation of the at least one modified viral envelope protein into the baculovirus particle upon virus production in insect cells; and (3) at least one heterologous expression cassette B containing a promoter and a nucleotide sequence coding for at least one heterologous gene product, the expression cassette enabling the expression of said gene product(s) in insect cells and incorporation of the gene product(s) into the baculovirus particle upon virus production in insect cells with the proviso that, if the gene product is a heterologous polypeptide it is an essentially non-fluorescent polypeptide, with the proviso that the heterologous polypeptide is optionally fused to a fluorescent label.

2. The vector system of claim 1 wherein the at least one heterologous gene product of the at least of heterologous expression cassette B is incorporated in the interior of the baculovirus particle.

3. The vector system of claim 1 or 2 wherein the at least one heterologous gene product of the at least of heterologous expression cassette B is non-covalently incorporated into the baculovirus particle.

4. The vector system according to any one of the preceding claims wherein the at least one heterologous expression cassette B contains a promotor and (i) an open reading frame coding for at least one heterologous essentially non-fluorescent polypeptide, optionally fused to a fluorescent label and/or (ii) a sequence coding for at least one heterologous RNA transcribed by the promoter. 1

5. The vector system according to any one of the preceding claims wherein further comprising at least one expression cassette C, optionally contained in the baculovirus genome (1), containing a promoter and nucleotide sequence coding for at least one heterologous gene product, the expression cassette enabling the expression and/or transcription of said gene product in mammalian cells.

6 The vector system of claim 5 wherein the at least one expression cassette C contains a promotor and (a) an open reading frame coding for at least one heterologous polypeptide and/or (b) a sequence coding for at least one heterologous RNA transcribed by the promoter, wherein the heterologous polypeptide and the heterologous RNA is/are expressed or transcribed, respectively, in mammalian cells.

7. The vector system according to any one of the preceding claims wherein the heterologous polypeptide and/or the heterologous RNA is/are modulators of a physiological process selected from the group consisting of intracellular organization, metabolism, responsiveness, immune system processes, cell signaling, movement, genome modifications, reproduction, or death, preferably cell death.

8. The vector system of claim 7 wherein the metabolism includes a cellular process selected from the group consisting of intracellular trafficking, RNA transcription, protein translation and cellular energy levels.

9. The vector system of claim 7 wherein the immune system process is selected from the group consisting of innate, cellular and adaptive immune system processes.

10. The vector system of claim 7 wherein the genome modification is selected from the group consisting of base substitutions, base deletions, base insertions, and epigenetic modifications of host cell DNA.

11. The vector system according to any one of the preceding claims wherein viral envelope protein is selected from the group consisting of VSV-G, HIV gp120, baboon envelope protein, and modified versions of baculovirus envelope protein GP64.

12. The vector system of claim 11 wherein the VSV-G comprises, essentially consisting of or consisting of the amino acid sequence of SEQ ID NO: 3 or a transcriptionally functional homolog thereof. 2

13. The vector system of claim 12 wherein the transcriptionally functional homolog has a sequence identity at least 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 % to SEQ ID NO: 3.

14. The vector system according to any one of the preceding claims wherein the element (2) comprises at least one further expression cassette containing a promoter and an open reading frame coding for an additional modified viral envelope protein comprising an immune complement system inhibitor, wherein the additional modified viral envelope protein is expressed in insect cells and incorporated into the baculovirus particle upon virus production in insect cells.

15. The vector system of claim 14 wherein the immune complement system inhibitor is selected from the group consisting of decay-accelerating factor (DAF), factor H (FH)- like protein-1 (FHL-1), C4b-binding protein (C4BP), and membrane cofactor protein (MCP).

16. The vector system of claim 14 or 15 wherein the immune complement system inhibitor is fused to a moiety selected from the group consisting of the baculovirus envelope protein GP64 and the membrane anchor of the vesicular stomatitis virus-G (VSV-G) protein.

17. The vector system of claim 16 wherein the protein GP64 comprises, essentially consist of or consists of the amino acid sequence of SEQ ID NO: 13.

18. The vector system of claim 16 wherein the membrane anchor comprises, essentially consists of or consists of the amino acid sequence of SEQ ID NO: 19.

19. The vector system of claim 16 or 17 wherein the complement system inhibitor fused to GP64 is a DAF-GP64 fusion protein.

20. The vector system of claim 19 wherein the DAF-GP64 fusion protein comprises, essentially consists of or consists of the amino acid sequence of SEQ ID NO: 15.

21. The vector system of claim 14 or 18 wherein the complement system inhibitor fused to the membrane anchor of VSV-G is a DAF-VSV-G membrane anchor fusion protein. 3

22. The vector system of claim 21 wherein the DAF-VSV-G membrane anchor fusion LU103239 protein comprises, essentially consists of or consists of the amino acid sequence of SEQ ID NO: 16.

23. The vector system according to any one of the preceding claims wherein the RNA produced by expression cassette(s) B comprises sequence elements that enable the RNA to be translated in mammalian cells.

24. The vector system of claim 23 wherein the sequence elements comprise at least one internal ribosomal entry site.

25. The vector system according to any one of the preceding claims wherein the expression cassette B codes for a heterologous polypeptide selected from the group consisting of apoptosis factors, iPSC reprogramming factors, tumor suppressor proteins/peptides, and vaccines.

26. The vector system of claim 25 wherein the apoptosis factor is selected from the group consisting of Apoptosis Inducing Factor, caspase-3, caspase-8, and caspase-9.

27. The vector system of claim 25 or 26 wherein the iPSC reprogramming factor is selected from the group consisting of Oct4 (Pou5f1), cMyc, Sox 2, KIf4, LIN28A, NANOG, L-MYC, GLIS1, ESRRB, UTF1, NR5A2, SALL4, PRDM14, DPPA2, DPPA4A, TDGF1, RARG, MBD3, NR2F2, ZIC3, ASCI1, LMX1A, MYT1L, BM2 AND NURR1.

28. The vector system according to any one of claims 25 to 27 wherein the tumor suppressor protein/peptide is selected from the group consisting of p53, pRB, BCL2, SWI/SNF, pVHL, APC, CD95, ST5, YPEL3, ST7, and ST14, p16, BRCA2, APC and NBD peptides.

29. The vector system of claim 28 wherein the tumor suppressor p53 comprises, essentially consists of or consists of the amino acid sequence of SEQ ID NO: 10 or a transcriptionally functional homologue thereof.

30. The vector system of claim 29 wherein the transcriptionally functional homologue has a sequence identity at least 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 % to SEQ ID NO: 10. 4

31. The vector system according to any one of claims 25 to 30 wherein the vaccine is LU103239 selected from the group consisting of protein vaccines, peptide vaccines, and RNA vaccines.

32. The vector system according to any one of claims 25 to 31 wherein the vaccine is selected from the group consisting of live-attenuated vaccines, inactivated vaccines, subunit, recombinant, polysaccharide, conjugate vaccines, and toxoid vaccines.

33. The vector system according to any one of the preceding claims wherein the expression cassette(s) B and/or C is/are coding for an RNA selected from the group consisting of an siRNA, a dsRNA, a shRNA, a miRNA, or a ribozyme. an siRNA, a dsRNA, a shRNA, a miRNA and a ribozyme.

34. The vector system according to any one of the preceding claims wherein expression cassette(s) B and/or C is/are coding for one or more gene products selected from the group consisting of a DNA endonuclease protein, a transposon, a DNA base editor, a prime editor, one or more protein and/or RNA components of a transposon-encoded CRISPR-Cas system, a Cas-Reverse Transcriptase fusion protein and a transposase protein.

35. The vector system of claim 34 wherein the endonuclease protein the group consisting of RNA-guided nucleases, TALENS, Zn Finger Nucleases, and homing endonucleases.

36. The vector system of claim 34 or 35 wherein the one or more elements of the transposon-encoded CRISPR-Cas system comprises Vibrio cholerae Tn6677 from Escherichia coli carrying transposon-associated molecular machineries, preferably including the DNA-targeting complex Cascade and the transposition protein TniQ.

37. The vector system of claim 35 or 36 wherein the RNA-guided nuclease is a selected from the group consisting of RNA-guided nucleases form CRISPR-Cas system types , II, I, IV, V and//or VI and DNA base editors.

38. The vector system of claim 37 wherein the RNA-guided nuclease is selected from the group consisting of Cas9, dCas9, SpCas9, Cas9 10a nickase, Cas 12b, PE1, PE2 and Cpft1. 5

39. The vector system of claim 38 wherein Cas9 comprises, essentially consists of or LU103239 consists of the amino acid sequence of SEQ ID NO: 6 or a transcriptionally functional homologue thereof.

40. The vector system of claim 39 wherein the transcriptionally functionally homologue has a sequence identity at least 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 % to SEQ ID NO: 6.

41. The vector system according to any one of claims 38 to 40 wherein dCas9 comprises, essentially consists of or consists of the amino acid sequence of SEQ ID NO: 7 or a transcriptionally functional homologue thereof.

42. The vector system of claim 41 wherein the transcriptionally functionally homologue has a sequence identity at least 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 % to SEQ ID NO: 7.

43. The vector system according to any one of claims 37 to 42 wherein PE1 comprises, essentially consists of or consists of the amino acid sequence of SEQ ID NO: 8 or a transcriptionally functional homologue thereof.

44. The vector system of claim 43 wherein the transcriptionally functionally homologue has a sequence identity at least 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 % to SEQ ID NO: 8.

465. The vector system according to any one of claims 37 to 44 wherein PE2 comprises, essentially consists of or consists of the amino acid sequence of SEQ ID NO: 9 or a transcriptionally functional homologue thereof.

46. The vector system of claim 45 wherein the transcriptionally functionally homologue has a sequence identity at least 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 % to SEQ ID NO: 9.

47. The vector system of claims 35 or 36 comprising at least one additional expression cassette B and/or C coding for at least one guide RNA.

48. The vector system of claim 47 wherein the guide RNA is selected from the group consisting of a single guide RNA (sgRNA) and a prime editing guide RNA (pegRNA). 6

49. The vector system of claim 34 wherein the transposon is selected from the group consisting of retrotransposons and DNA transposons.

50. The vector system of claim 49 wherein the transposon is selected from the group consisting of Transposon TN3, Transposon TN10, Transposon Tn6677, and Transposon TN7.

51. The vector system of claim 50 wherein the Transposon TN7 comprises or consists of genes tnsA, tnsB, tnsC, and tnsD.

52. The vector system of claim 50 or 51 wherein Transposon TN7 additionally encodes an RNA-guided nuclease.

53. The vector system according to any one of claims 34 to 52 comprising at least one expression cassette C as defined in claim 3 or 4 comprising one or more recognition motifs of a protein selected from the group consisting of a DNA endonuclease protein, a DNA base editor, a prime editor, a transposon-encoded CRISPR-Cas system, a Cas-Reverse Transcriptase fusion protein and a transposase protein, enabling said one or more recognition motifs being bound, modified and//or cleaved by said protein.

54. The vector system according to any one of claim 34 to 53 wherein the DNA endonuclease protein, the DNA base editor, the prime editor, the transposon-encoded CRISPR-Cas system, the Cas-Reverse Transcriptase fusion protein and/or the transposase protein are selected such that said DNA endonuclease protein, DNA base editor, prime editor, transposon-encoded CRISPR-Cas system, Cas-Reverse Transcriptase fusion protein and/or transposase protein bind(s), modifies/modify and/or cleave(s) one or more recognition motifs present in a mammalian cell.

55. The vector system of claim 54 wherein the at least one expression cassette C of claim 3 or 4 is selected such that it becomes permanently integrated into a mammalian cell by an endogenous mammalian DNA repair system and/or by said DNA endonuclease protein, DNA base editor, prime editor, transposon-encoded CRISPR-Cas system, Cas-Reverse Transcriptase fusion protein and/or transposase protein. 7

56. The vector system according to any one of the preceding claims wherein the total LU103239 combined size of heterologous expression cassettes A, B, and, optionally, C is more than 7,500 bp, preferably more than 10,000 bp, more preferably 15,000 bp, even more preferably more than 20,000 bp.

57. The vector system according to any one of the preceding claims wherein the promoter(s) of expression cassette(s) is selected from the group consisting of a Polyhedrin promotor and a p10 promoter.

58. The vector system of claim 57 wherein the promoter comprises, essentially consists of or consists of the nucleotide sequence of SEQ ID NO: 1.

59. The vector system of claim 57 wherein the promoter comprises, essentially consists of or consists of the nucleotide sequence of SEQ ID NO: 2.

60. The vector system according to any one of the preceding claims wherein the baculovirus genome is derived from a nuclear polyhedrosis virus (NPV).

61. The vector system of claim 60 wherein the nuclear polyhedrosis virus is selected from the group consisting of the multiple nucleocapsids per envelope (MNPV) subgenera of the NPV genera of the Eubaculovirinae subfamily (occluded Baculoviruses) of the Baculoviridae family of insect viruses.

62. The vector system of claim 60 or 61 wherein the NPV is an Autographa californica nuclear polyhedrosis virus (ACMNPYV).

63. The baculovirus vector system according to any one of the preceding claims wherein expression cassette(s) A and/or B is/are contained in a transfer vector suitable for fusion with genetically modified baculovirus DNA, in a genetically modified baculovirus DNA, in a separate chromosomal DNA within insect cells infected with a baculovirus, or in a non-chromosomal DNA within insect cells infected with a baculovirus.

64. Composite baculovirus DNA comprising the baculovirus vector system of any one of claims 1 to 62. 8

65. A host cell comprising the baculovirus vector system according to any one of claims 1 LU103239 to 62 and/or the composite baculovirus DNA of claim 64.

66. The host cell of claim 65 wherein the host cell is selected from the group consisting of bacteria, yeast and insect cells.

67. The host cell of claim 65 or 66 wherein the host cell is an insect cell comprising the composite baculovirus DNA of claim 62.

68. The host cell of claim of claim 66 or 67 wherein the insect cell is selected from the group consisting of insect cells derived from Spodoptera frugiperda, Trichoplusia ni, Plutella xylostella, Manduca sexta, and Mamestra brassicae.

69. The host cell of claim 66 or 67 wherein the insect cell is an IPLB-SF21AE cell or its clonal isolate Sf9.

70. A cell culture comprising the host cell according to any one of claims 65 to 69 in a cell culture medium adapted for storage and/or propagation of the host cell.

71. A recombinant baculovirus particle comprising the composite baculovirus DNA of claim 64.

72. A recombinant baculovirus particle comprising element (1) as defined in claim 1, at least one modified viral envelope protein expressed from the heterologous expression cassette A as defined in claim 1, at least one heterologous gene product expressed from the at least one heterologous expression cassette B as defined in claim 1, and, optionally, at least one heterologous gene product expressed from the at least one heterologous expression cassette C as defined in claim 1.

73. A pharmaceutical composition comprising the host cell according to any one of claims 65 to 69 or the cell culture of claim 68 or the recombinant baculovirus particle of claim 69 or 70, preferably in combination with at least one pharmaceutically acceptable excipient and/or carrier and/or diluent.

74. Use of the baculovirus vector system according to any one of claims 1 to 62, the composite baculovirus DNA of claim 64, the host cell according to any one of claims 65 to 69, the cell culture of claim 70, the recombinant baculovirus particle of claim 71 9 or 72 or the pharmaceutical composition of claim 73, in gene therapy, gene editing, ~~ LU103239 gene engineering, cell therapy, immunotherapy, delivery of RNA interference systems, delivery of apoptosis factors, iPS reprogramming, synthetic biology, production of protein complexes, modification of cell signaling, drug discovery, diagnostics, and vaccines.

75. A method for delivering DNA together with one or more heterologous gene products into a mammalian cell, mammalian tissue, mammalian organ and/or mammalian organism comprising the steps of: (a) introducing the baculovirus vector system of claims 1 to 62 or the composite baculovirus DNA of claim 64 into insect cells as defined in any one of claims 66 to 69, (b) culturing said insect cell under conditions allowing the expression of the gene product of expression cassette B and, optionally, expression cassette C, and allowing the production of recombinant baculovirus particles; (c) harvesting the baculovirus particles produced in step (b); and (d) transducing the mammalian cell, tissue, organ, and/or organism with the baculovirus particles harvested in step (0).

76. The method of clam 75 wherein wherein said culture conditions are maintained until the number of insect cells is between about 10° and 10" and/or until the number of baculovirus particles is between 10% and 10".

77. The method of claim 75 or 76 wherein the mammalian cell is selected from the group consisting of a primary cell, an immortal cell line, a cell being part of a tissue and a cell being part of an organ from a living mammal.

78. The method of claim 77 wherein the primary cell is selected from the group consisting of a primary immune cell, a neuronal cell, an adipose cells, a bladder cell, a blood vessel cell, a cardiac cell, a cartilage cell, a bone cell, a bone marrow cell, a bronchial/tracheal cell, a cardiac cell, a colon cell, a dermal cell, an epidermal cell, an esophagus cell, a gallbladder cell, a gastrointestinal cell, a hepatic cell, a keratinocyte, a lung cell, a lymphatic cell, a mammary cell, an ocular cells, a pancreatic cell, an iPS cell, a fertilized oocyte and an unfertilized oocyte. 10

79. The method of claim 78 wherein the primary immune cell is selected from the group ~~ LU103239 consisting of a B cell, a T cell, a Dendritic cell, a TIL, an activated T cell, and a resting T cell.

80. The method of claim 75 or 77 wherein the organ is selected from the group consisting of an artery, bone, bone marrow, brain, gallbladder, heart, intestines, kidney, larynx, liver, lung, lymph node, muscles, ovary, pharynx, placenta, prostate, skin, a spleen, stomach, thyroid gland, urethra, urinary bladder, and a vein. 11