Improving DNA Vaccine Performance Through Vector ...

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Improving DNA Vaccine Performance Through Vector Design James A. Williams* Nature Technology Corporation/Suite 103, 4701 Innovation Drive, Lincoln, NE 68521, USA Abstract: DNA vaccines are a rapidly deployed next generation vaccination platform for treatment of human and animal disease. DNA delivery devices, such as electroporation and needle free jet injectors, are used to increase gene transfer. This results in higher antigen expression which correlates with improved humoral and cellular immunity in humans and animals. This review highlights recent vector and transgene design innovations that improve DNA vaccine performance. These new vectors improve antigen expression, increase plasmid manufacturing yield and quality in bioreactors, and eliminate antibiotic selection and other potential safety issues. A flowchart for designing synthetic antigen transgenes, combining antigen targeting, codon-optimization and bioinformatics, is presented. Application of improved vectors, of antibiotic free plasmid production, and cost effective manufacturing technologies will be critical to ensure safety, efficacy, and economically viable manufacturing of DNA vaccines currently under development for infectious disease, cancer, autoimmunity, immunotolerance and allergy indications.

Keywords: Antibiotic-free, DNA vaccination, fermentation, non-viral, plasmid, vector. 1. INTRODUCTION DNA vaccines are modified bacterial plasmids that combine a bacterial region that encodes genes necessary for selection and propagation in Escherichia coli with a eukaryotic region that encodes a transgene and sequences required to express the transgene in the target organism Fig. (1A). The vector is transfected into target tissue cells during delivery to the recipient. Upon entry to the cell nucleus the vector transcribes the encoded transgene antigen into mRNA which is exported to the cytoplasm and translated into antigen protein. If the vector transfects an Antigen Presenting Cell (APC), host-expressed antigen may be directly presented to CD8 and CD4 T-cells by major histocompatibility complex (MHC) class I or II respectively (Direct route) Fig. (1B). If the vector transfected a non-APC, antigen presentation may be by cross presentation (Indirect route) Fig. (1B). Transfected DNA mediated activation of innate immunity is a critical danger signal necessary to induce adaptive immune response to the MHC presented antigen [1-3]. DNA vaccines activate a number of intracellular DNA sensing pathways (reviewed in [4-6]). DNA vaccination “adjuvant effect” is primarily mediated in a sequence non -specific manner by B DNA (the prevalent double helical DNA form) mediated activation of the cytoplasmic double stranded DNA sensing stimulator of interferon genes/TANK-binding kinase 1 (STING/TBK1) dependent innate immune signaling pathway (reviewed in [4-7]). Recently it has been determined that B DNA is recognized by cyclic-GMP-AMP (cGAMP) synthase (cGAS) [8] which produces cGAMP that activates STING to induce type 1 interferon and NF-B production. This cytoplasmic pathway is necessary to induce antigen specific *Address correspondence to this author at the Nature Technology Corporation/Suite 103, 4701 Innovation Drive, Lincoln, NE 68521, USA; Tel: +1-402-323-6289; Fax: +1-402-323-6292; E-mail: [email protected] 1566-5232/14 $58.00+.00

B cells and CD4+ and CD8+ T-cells in response to IM DNA vaccination with EP [9]. However, at least with IM DNA vaccination without EP, the endosomal CpG DNA motif sensing Toll-like receptor 9 (TLR9) signaling pathway plays a role in priming CD8+ T cell responses [10, 11]. The importance of TLR9 to DNA vaccination may be delivery and tissue specific. For example, cationic liposome mediated plasmid delivery to the lung activates a CpG dependent inflammation response [12]. Cytoplasmic DNA may also trigger interleukin1 production through activation of the absence in melanoma 2 (AIM2) inflammasome [13]. It remains to be established if inflammasome activation contributes to DNA vaccination. DNA vaccines have an excellent safety profile with no reported safety issues such as autoimmunity or host genome insertional mutagenesis from numerous human clinical trials (reviewed in [1-3]). Additionally, DNA vaccines do not induce anti-vector immunity as occurs with viral vector particles. This is a key advantage of DNA vaccination since they can be utilized in multiple boosts or with multiple products. DNA vaccination of neonate macaques is effective, even if maternally derived neutralizing antibodies are present [14]. Further, DNA vaccine manufacture is much faster, less complex, and scalable than viral vector platforms and plasmid DNA is exceptionally stable [15]. Four DNA vaccines have been licensed for animal health applications including a therapeutic cancer vaccine for dog melanoma, a gene therapy to improve breeding pig sows litter survival, and prophylactic vaccines for infectious haematopoietic necrosis virus in fish and West Nile virus in horses [1]. While no human DNA vaccines have been licensed, more than 200 DNA vaccine intervention human clinical trials on a wide spectrum of prophylactic or therapeutic vaccines for infectious disease or cancer applications are listed as completed or ongoing (http://www.clinicaltrials.gov). © 2014 Bentham Science Publishers

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Fig. (1): DNA Vaccine composition and mechanism A. Composition. Vectors combine a bacterial region including sequences required for selection and propagation in E. coli with a eukaryotic region including sequences required for antigen expression in the target organism. B. Mechanism. Formulated plasmid delivered to a target organism. A delivery complex may be used but naked DNA is typically utilized with electroporation (EP) or needle free jet delivery that increase plasmid transfer to the cytoplasm. During delivery plasmid transfects either NonAPC (left) or APC (right) and, after trafficking to the nucleus, expresses antigen encoding mRNA which is exported from the nucleus and translated into antigen protein. Transgenes can be routed for secretion, proteosomal degradation, or endosomal processing during translation using targeting peptide sequences. Antigen is presented by MHC I or MHC II either directly in the case of APC cell transfection, or indirectly on APC MHC I by cross presentation of cell associated exogenous antigen or on APC MHC II after endolytic processing of captured secreted antigen. Extracellular antigen may also be captured by B cells to activate a humoral response that also requires antigen activated CD4 T helper cell produced cytokines.

DNA vaccine priming combined with a protein or viral vector heterologous boost has been shown to improve immune response with multiple antigens [2, 16-18]. For example, influenza hemagglutinin antigen DNA primeheterologous inactivated vaccine protein boost vaccination induces broadly cross neutralizing antibodies [16, 19-21]. Alternatively, in some models simultaneous administration of protein and DNA vaccine may be used to induce tolerance and not immunity (see Section 2.6).

forms such as EP [22-24], needle free jet-injection [25-27] and liposome [28] have been developed that improve plasmid transfection, transgene expression, and immunity. One of the reasons DNA vaccination is more difficult in humans relative to mice may be that human serum amyloid P binds plasmid more strongly than the murine cognate. Indeed, DNA vaccine induced adaptive immune responses are decreased in transgenic mice expressing this human serum protein [29, 30].

However, for DNA only vaccination, it is more difficult to obtain acceptable efficacy in humans and large animals than in mice. To overcome this obstacle, DNA delivery plat-

Most delivery platforms promote plasmid transfer across the cell plasma membrane barrier to directly (EP, jet injection) or indirectly (liposome) transfect plasmid into the cell

Improving DNA Vaccine Performance Through Vector Design

cytoplasm but do not deliver DNA directly to the nucleus [31]. Smaller vectors are more compact, which improves vector transfection and diffusion through the cytoplasm to the nucleus. Nuclear plasmid import may be increased using nuclear targeting sequences [32]. Transgene expression occurs after nuclear import; optimization of the bacterial backbone and eukaryotic regions can dramatically increase transgene expression. In this review, vector design innovations that improve DNA vaccine efficacy, safety and manufacturing are discussed. Critical production issues that need to be addressed prior to cGMP manufacture of plasmid for clinical trials are discussed. 2. VECTOR DESIGN DNA vaccine vectors combine a bacterial region that provides propagation and selection in the Escherichia coli (E. coli) host with a eukaryotic region that directs transgene expression in the target organism Fig. (1A). The bacterial region encodes a selectable marker and a high copy replication origin, typically the pUC origin. The eukaryotic region contains a promoter-gene of interest-polyadenylation signal (polyA) expression cassette. After the plasmid enters the nucleus, the promoter transcribes the transgene into an mRNA that is cleaved, polyadenylated and exported to the cytoplasm. The transgene ATG start codon is within a Kozak sequence (gccgccRccATGG consensus, critical residues in caps, ATG start codon is underlined, R = A or G) that directs efficient transgene translation by ribosomes. The 3' and 5' untranslated regions (UTRs) of the mRNA should not contain open reading frames (ORFs) since these may be translated and evoke an adaptive immune response [33]. The most common promoter used in DNA vaccines is the human Cytomegalovirus (CMV) promoter since it is highly active and constitutively transcribes higher levels of mRNA than alternative cellular or viral promoters. PolyA signals typically are derived from either the bovine growth hormone or rabbit globin genes that contain polyadenylation efficiency enhancing accessory sequences flanking the polyadenylation site (AATAAA). A critical part of vector design is careful selection and assembly of bacterial region selection and replication sequences since bacterial region sequences can dramatically impact plasmid quality and production yield in the E. coli host and transgene expression in the target organism [34, 35]. Transgene expression inhibition may be due to double stranded RNA (dsRNA) production from vector backbone encoded cryptic promoters. dsRNA may inhibit expression through RNA interference or by protein kinase R (PKR) mediated translational inhibition [36-38]. First generation DNA vaccine vectors such as gWIZ (Genlantis, San Diego, CA, USA) and pVAX1 (Invitrogen, Carlsbad, CA, USA) utilize the kanamycin resistance selectable marker (kanR). The gWIZ vector, which encodes an extensively optimized bacterial region [34] as well as an intron, has 5-fold improved transgene expression and higher manufacturing yields than pVAX1 [39]. In pVAX1 the cryptic eukaryotic promoter within the pUC origin [36] is antisense to the transgene; dsRNA may be produced which

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could reduce transgene expression through PKR mediated translational inhibition or RNA interference. pVAX1 transgene expression is also reduced compared to gWIZ by bacterial region encoded inhibitory sequences (see Section 2.3). 2.1. Vector Design Considerations Design criteria for regulatory agency compliant new generation vectors that combine superior transgene expression with improved manufacturing yields are outlined below. See [24, 40-46] for detailed reviews of transgene and DNA vaccine vector design. Regulatory: Guidance documents published by the European Union (EU) and FDA provide insight into regulatory agency concerns regarding human plasmid DNA gene medicines [47-49]. Vector encoded cryptic peptides within the 5’ and 3’ UTRs if translated in addition to the transgene [50] may cause an inappropriate adaptive immune response [33, 51]. Another concern is sequences within vector backbones that encode ORFs that may be expressed in the target organism, for example antibiotic resistance markers. The EU recommends removing kanR selection markers from vectors [49] (see Section 2.3). Interestingly, retrofit of the kanR marker with short RNA markers for antibiotic-free selection unexpectedly also improves transgene expression (see Section 2.3). Expression: a 50-150 bp 5' UTR with no additional ATG start codons is upstream of the authentic ATG containing Kozak sequence. The UTR should not contain Kozak sequence binding stable mRNA secondary structures that might interfere with Kozak sequence mediated ribosome recruitment. Transgene expression is increased by including an intron [2]. Splicing enhancers within the intron and the flanking exons can increase transgene expression through increased intron splicing [52-54]. Some UTRs can improve translation efficiency. For example, the human T-cell leukemia virus type I R (HTLV-I R) region encoded within the 5' UTR increases mRNA translation and transgene expression in mice and nonhuman primates [50, 55]. HTLV-I R encoding DNA vaccines have been established as safe in nonclinical toxicology and numerous human clinical trials [20]. The bacterial region should not contain antisense oriented cryptic eukaryotic promoters since dsRNA may promote RNA interference mediated transgene silencing [37]. Evaluation of expression level differences between vectors in vitro must be done using transfections of limiting amounts of plasmid DNA into mammalian cells since transgene mRNA levels can easily saturate in vitro protein production capacity [56]. Interestingly, reduction of the bacterial region to less than 500 bp unexpectedly improves transgene expression (see Section 2.5). Manufacture/quality: Vectors should be optimized for production quality and yield to facilitate cost effective manufacture post-licensure. A plasmid should be stable and not undergo nicking, multimerization, or sequence deletion or rearrangement during fermentation and nicking or irreversible denaturation during cell lysis and purification. Bioinformatics can be used to identify problematic sequences in a vector (or gene insert, see Section 2.8) [40, 44]. For example, certain DNA sequences such as direct or inverted repeats, palindromes or extended homopurine-homopyrimidine tracts


are unstable and prone to deletion or rearrangement [43]. Large palindromes may also inhibit plasmid replication reducing copy number and quality. Chi sites increase plasmid multimerization [40, 41, 43]. Plasmid nicking often occurs at local single stranded regions formed in AT-rich sequences and cruciforms. Z DNA or triplex forming sequences has been shown to reduce plasmid quality [57]. Often vector sequences upstream of the transgene encode cryptic bacterial promoters that mediate unintended transgene expression in the bacterial production strain. This often destabilizes the plasmid due to transgene toxicity [40], necessitating vector redesign to prevent transgene translation in the bacterial host. For regulatory reasons investigators often do not want to alter vectors between phases of human clinical trial evaluation. In these cases transgene-complementary RNA, expressed from the genome of an engineered designer host strain [58], can be used to improve plasmid stability and yield by antisense inhibition of toxic transgene translation. 2.2. Vector Modifications to Increase Antigen-Specific Adaptive Immunity A variety of approaches to increase plasmid DNA vaccine efficacy have been developed. Many of these are design innovations increase activation of innate immunity resulting in improved adaptive immunity. Several strategies are summarized below. Numerous adjuvant protein plasmids have been developed that improve immune response to codelivered DNA vaccine plasmid antigens. Adjuvant plasmids encode cytokines (e.g., interleukin-12)[59], costimulatory molecules (e.g., CD40), chemokines (e.g., RANTES), (reviewed in [24, 60, 61]) or innate immune response signaling molecules [e.g., interferon regulatory factor-3 (IRF3), TLR or cytoplasmic adaptor molecules] (reviewed in [6, 62]). For cancer therapy, adjuvant plasmids may be utilized alone (e.g., interleukin-12 for melanoma; [63]) or in combination with tumor antigen encoding DNA vaccines since tumor antigens are not inherently immunogenic [64]. Alternatively, a DNA vaccine vector may be modified to incorporate a short hairpin RNA (shRNA) as shown in Fig. (1A) that encodes an RNA that silences expression of an immunosuppressive molecule [65], for example antigenpresentation attenuators [66] such as suppressor of cytokine signaling (SOCS) 1 [67], proapoptotic signaling proteins such as Fas ligand [68] or furin convertase to downregulate immunosuppressive transforming growth factors 1 and 2 [69]. The composition and positioning of the shRNA transcription unit needs to be carefully designed since alternations to the bacterial region can dramatically impact expression [34, 35] and insertion within an intron of the vaccine antigen transcription unit may affect intron splicing accuracy or efficiency. Alternatively, a microRNA sequence, without a promoter, can be encoded within the vaccine intron transcription unit. The intronic microRNA is expressed from the vaccine antigen promoter and functional microRNA excised from the intron during splicing [70]. Another approach is to add DNA or RNA adjuvants to the vector backbone (plasmid backbone adjuvant). These adjuvant plasmids avoid the autoimmunity concerns associated with adjuvant protein plasmids since the backbone en-

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coded DNA or RNA adjuvant will not be the target of adaptive immunity Vector backbones are modified to encode DNA based adjuvants by addition of immunostimulatory six base pair CpG motif (XXCGXX) agonists of the endosomal TLR9 innate immune receptor. TLR9 activating CpG motifs are used since the cytoplasmic DNA sensing pathways are sequence nonspecific. [71] Care must be taken in CpG motif design since many CpG motifs are immunosuppressive and the optimal immunostimulatory CpG agonists are species specific. For example GTCGTT is optimal for human TLR9 while GACGTT is optimal for murine TLR9 [72]. While variable results have been obtained, incorporating additional CpG motifs has in some cases improved DNA vaccination immune responses [73, 74]. Addition of CpG motifs to a vector backbone may inadvertently affect transgene expression (see Section 2.3) which may account for the variable success of this approach. As well, differences in endosomal trafficking between delivery modalities may alter the efficiency of endosomal TLR9 activation by DNA vaccine encoded CpG motifs. Vector backbones are modified to encode RNA based adjuvants by engineering to coexpress immunostimulatory RNA (isRNA) with antigen. Two approaches are used. For RNA Pol II transcription isRNA is encoded either in the 3' UTR downstream of the transgene or in a second transcription unit within the vector backbone. For RNA Pol III transcription isRNA is transcribed from an independent transcription unit either in the vector backbone or nested within an intron Fig. (1A). DNA vaccination with vectors encoding RNA Pol II or RNA Pol III expressed isRNA improved antigen-specific humoral and/or cellular response [75-77]. This may be through activation of retinoic acid inducible gene-1 (RIG-I) signaling (RNA Pol III; [76]) the activation of which has demonstrated adjuvant properties [78] or alternative signaling pathways responsive to RNA ligands [79] that combine with vector DNA mediated signaling to create unique polyvalent innate immune pathway activation [80]. 2.3. Antibiotic-Free Selection Using RNA Selection Markers High-copy ColE1-type plasmids tend to be excluded from the nucleoid and clustered at the cell poles so random partitioning to the two daughter cells during cell division is efficient [81]. However, rare cells that have lost plasmid may have a growth advantage over plasmid containing cells. Therefore a plasmid selection marker is included in DNA vaccine vectors. Ampicillin resistance markers are not generally acceptable due to potential for hyper reactivity to residual trace lactam antibiotic in the DNA vaccine. Regulatory agency guidance’s recommend removing antibiotic resistance markers from DNA vaccine vectors. Environmental contamination with antibiotics used in manufacture or plasmid borne antibiotic resistance markers [82] is a concern. As well, a patient’s endogenous microbial flora may take up the antibiotic resistance marker during treatment (e.g., topically applied plasmid DNA transfecting resident skin microorganisms). Alternatively, the selection marker may be incorporated into the cellular genome after treatment and the antibiotic resistance gene expressed from a host cell promoter ad-

Improving DNA Vaccine Performance Through Vector Design

jacent to the inserted marker. The European Pharmacopoeia states “Unless otherwise justified and authorized, antibioticresistance genes used as selectable genetic markers, particularly for clinically useful antibiotics, are not included in the vector construct. Other selection techniques for the recombinant plasmid are preferred” [83]. The European Medicines Agency (EMA) determined that neomycin and kanamycin, due to current use in critical clinical settings, cannot be classified as having minor therapeutic relevance [49]. Alternative non-antibiotic selection markers are needed that address these regulatory concerns. As with antibiotic resistance markers, all protein selection markers have the risk of unintentional expression in the patient. Several non-protein antibiotic-free (AF) selection markers have been developed [40, 84, 85]. Of these vectors encoding RNA based antibiotic-free selection markers have superior expression and manufacture. For example RNA based selection markers include: RNA-OUT, a small 70 bp antisense RNA (e.g. the NTC868 series sucrose selection vectors) Fig. (2) [86]; a nonsense suppressor tRNA (e.g. the pCOR and pFAR4 vectors) [87, 88]; ColE1 origin-encoded RNAI antisense RNA (e.g. pMINI vector) [89, 90]. With these vectors the RNA marker maintains plasmid retention through regulation of the translation of a selectable marker expressed from the host chromosome Fig. (2). RNA selection markers are compatible with high yield fermentation manufacture, for example, plasmid yields of 1,800 mg/L with RNA-OUT vectors [91] and 900 mg/L with pMINI vectors [92]. Plasmids using antibiotic-free RNA selection markers have improved transgene expression compared to the equivalent kanR antibiotic selection marker vectors. Thus removal of antibiotic selection markers to address regulatory concern has the unexpected benefit of improved performance. Several bacterial sequences that inhibit transgene expression have been identified [reviewed in (40]. For example, pro-

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moter expression is decreased by a transcriptional silencer in the TN5 kanamycin/neomycin resistance marker [94]. The pVAX1 vector contains this TN5 kanR marker in a 1,970 bp bacterial region. When the pVAX1 kanR gene is retrofitted with antibiotic-free RNA selection markers the resultant vectors have dramatically increased expression. For example, higher expressing vectors were obtained after removal of the pVAX1 kanR marker and selection using the pUC origin encoded RNAI RNA selection marker; (pMINI, 734 bp bacterial region) [95] or retrofit of the kanR marker with either RNA-OUT (pVAX1-AF, 1,195 bp bacterial region) [39] or an amber suppressor tRNA (pFAR4, 1,040 bp bacterial region) [88] RNA selection marker. Alternatively, improved expression with pVAX1 retrofitted with RNA selection markers may be due to the reduced vector size. While the transgene encoding retrofitted vectors are >2,000 bp which should have similar cytoplasmic mobility as the larger parental kanR plasmids [96] smaller vectors have higher transgene expression due to more effective cell transfection [97, 98]. An additional advantage is these smaller vectors are also more resistant to shear forces associated with delivery. For example Biojector mediated needle free delivery through mouse skin nicked 23.4% of a 3579 bp plasmid, but only 8.6 % of a 1900 bp vector [99]. 2.4. Vector Modifications to Improve Plasmid Expression Duration In certain tissues (e.g., liver) bacterial regions of approximately 1,000 bp or larger mediate transgene silencing, while sustained transgene expression is obtained with minicircle vectors containing shorter spacer regions (sequences linking the 5’ and 3’ ends of the eukaryotic region) of 500 bp or less [100]. Nontranscribed spacer region sequences may promote the formation of inhibitory chromatin which spreads to the eukaryotic region resulting in transcriptional silencing [101]. DNA vaccine vectors with short

Fig. (2): Sucrose selection with RNA-OUT selection marker encoding Nature Technology Corporation (NTC) NTC868 series targeting DNA vaccine plasmids. Left: Sucrose selection. NTC868 series plasmid borne 70 bp RNA-OUT antisense RNA hybridizes to a RNA-IN target sequence in the leader of a constitutively expressed mRNA. This inhibits levansucrase (sacB) translation, allowing survival in sucrose media. Right: NTC868 series targeting vectors. The pUC replication origin is the purple arrow while the RNA-OUT selection marker is the brown arrow. The CMV promoter, transgene and rabbit globin polyA are depicted with orange arrow, blue arrow and green box, respectively. The transgene is shown, with the insertion point for the N-terminal secretion (NTC8682) proteasome (NTC8684) or endosome (NTC8681) targeting peptides indicated. Republished with permission of Humana Press from Luke et al. 2014 [93]


spacer regions less than 500 bp may have increased duration antigen expression. Long spacer region vectors can be modified to increase antigen expression duration, for example by spacer region transcription in eukaryotic cells using a heterologous promoter [102] or flanking the eukaryotic region with cis-acting sequences that prevent bacterial region mediated promoter shutdown, such as matrix attachment regions (MARs)[103], insulators (e.g. cHS4, tRNA), [104] or ubiquitous chromation-opening elements (UCOEs)[105]. A drawback of these modified vectors is that the increased size reduces transfection efficiency and vector potency resulting in reduced antigen expression level. As well, some protective sequences, such as MARs, associate with chromatin and increase the frequency of undesirable genome integration [106]. One potential application of vectors with sustained antigen expression is to improve memory CD8+ T-cell maintenance [107-109]. Interestingly, compared to plasmid vectors, persistent expression minicircle vectors elicit superior CD8+ T cell responses [110]. Further, minicircle DNA delivered with EP had improved cellular and humoral immune responses compared to a plasmid control [111]. These results are suggestive that extended duration expression may be beneficial in the context of DNA vaccination. Minicircle vectors production processes are very inefficient rendering them not practical for commercial DNA vaccine applications. Minicircle vectors are derived from plasmid vectors via the action of phage recombinases on plasmid encoded recognition sequences to create circularized bacterial and eukaryotic regions (minicircle) which then must be separated during minicircle purification. Optimal reported yields are only 5 mg minicircle per liter culture [112]. 2.5. Vector Modifications that Improve Plasmid Expression Duration and Expression Level Transgene expression is limiting with DNA vaccination in large animals. Therefore vector and delivery innovations

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that increase transgene expression may improve adaptive immunity. Recently spacer region minimalization has been shown to dramatically improved vector expression level and duration. The pMINI, pFAR4 and NTC868 series RNA selection marker vectors (see Section 2.3) spacer region is coincident with the bacterial region in these vectors. These vectors encode the high copy number pUC replication origin which can be reduced to 700 bp without loss of high copy number replication. Further reduction of bacterial/spacer region size to