Hybrid Promoter Research Articles

In-silico construction of potential cis-acting elements from PD_CbNPR1 with T7 minimal promoter

A hybrid promoter is a synthetic promoter engineering technique that has a function as a tool for cis-acting elements validation. The hybrid promoter is assembled by combining the minimal promoter [MP] with fragments containing cis-acting elements. PD_CbNPR1 from Capsicum annuum cv. Berangkai has several potential cis-acting elements for the regulation of plant resistance gene expression but has not been validated in the in-vitro assay. The potential elements are the W-Box motif which has three variations, then the WLE1 and RAV1AAT which have one variation motif. Sequences containing the cis-acting elements of PD_CbNPR1 were synthesized as fragment g-block. The MP used was the T7 Promoter in the pET28a[+] plasmid. The g-block fragment will be fused in the upstream region of the T7 Promoter. Before constructing the hybrid promoter in-vitro, it is necessary to construct the T7 promoter with cis-acting elements in-silico. The software used in this research was the plasmid editor ApE vers.2.0.36. The in-silico construct of the T7 promoter with cis-acting elements aimed to obtain plasmid construction designs that will be assembled in-vitro. The constructs obtained in this study consisted of six variations, in the form of a circular plasmid contains the potential cis-acting element. These constructs are called constructs A [WBox1-WBox2-WBox3-WLE1-RAV1AAT + pET28a[+]], B [WBox1 + pET28a[+]], C [WBox2 + pET28a[+]], D [WBox3 + pET28a[+]], E [WLE1 + pET28a[+]], and F [RAV1AAT + pET28a[+]]. The six constructs will be used in further study to regulate gene expression, as a tool to validate the cis-acting elements’ role in-vitro.

Development of efficient synthetic promoters derived from pararetrovirus suitable for translational research.

In this study, useful hybrid promoters were developed for efficient ectopic gene expression in monocot and dicot plants, and they hold strong prominence in both transgenic research and biotech industries. This study deals with developing novel synthetic promoters derived from Rice Tungro Bacilliform Virus (RTBV) and Mirabilis Mosaic Virus (MMV). Despite numerous availability, there is a severe scarcity of promoters universally suitable for monocot and dicot plants. Here, eight chimeric promoter constructs were synthesized as gBlocks gene fragments through domain swapping and hybridization by incorporating important domains of previously characterized RTBV and MMV promoters. The developed promoter constructs were assessed for transient GUS expression in tobacco protoplast (Xanthi Brad) and agro-infiltrated tobacco, petunia, rice and pearl millet. Protoplast expression analysis showed that two promoter constructs, namely pUPMA-RP1-MP1GUS and pUPMA-RP4-MP1GUS exhibited 3.56 and 2.5 times higher activities than that of the CaMV35S promoter. We had observed the similar type of expression patterns of these promoters in agroinfiltration-based transient studies. RP1-MP1 and RP4-MP1 promoters exhibited 1.87- and 1.68-fold increase expression in transgenic tobacco plants; while, a 1.95-fold increase was found in RP1-MP1 transgenic rice plants when compared their activities with CaMV35S promoter. Furthermore, on evaluating these promoter constructs for their expression in the bacterial system, pUPMA-RP1-MP1GFP was found to have the highest GFP expression. Moreover, the promoter construct was also evaluated for its capacity to express the HMP3 gene. Biobeads of encapsulated bacterial cells expressing HMP3 gene under control of the pUPMA-RP4-MP1 promoter were found to reduce 72.9% copper and 29.2% zinc concentration from wastewater. Our results had demonstrated that the developed promoter constructs could be used for translational research in dicot, monocot plants and bacterial systems for efficient gene expression.

Combination of ARE and HRE cis- Regulatory Elements Elevates the Activity of Tumor-Specific hTERT Promoter

Tumor-specific promoters and cis-regulatory genetic elements are used for transcriptional control of therapeutic transgene expression in cancer gene therapy. HRE (hypoxia response element) and ARE (anti-oxidant response elements) cis-regulatory elements are targets for HIF1 and Nrf2 transcriptional factors, respectively, and mediate activation of gene transcription in a response to hypoxia and oxidative stress, characteristic of most solid tumors. Due to these features HREs and AREs are used in genetic constructs for cancer gene therapy to provide tumor-specific therapeutic transgene expression or replication of oncolytic adenovi-ruses. In this work on the basis of the tumor-specific promoter hTERT we have constructed hybrid promoters carrying combinations of HRE and ARE. We showed that upon imitation of hypoxia in human lung cancer cell lines the activity of the hybrid promoter HRE-ARE-hTERT is substantially higher compared to promoters carrying only ARE or HRE. Using in vitro suicide cancer gene therapy with the CD: UPRT/5-FC (cytosine deaminase; uracil phosphoribosyl transferase/5-fluorocytosine) enzyme-prodrug system as a model we showed an enhancement of the cytotoxic effect on human lung cancer cells upon imitation of hypoxia when cytosine deaminase: uracil phosphoribosyl transferase was expressed under the control of the HRE-ARE-hTERT promoter compared to HRE-hTERT and ARE-hTERT promoters. The novel hybrid promoter HRE-ARE-hTERT could be used for transcriptional targeting of therapeutic transgene expression or oncolytic adenovirus replication upon development of novel anti-cancer gene therapeutics.

Engineered miniature H1 promoters with dedicated RNA polymerase II or III activity

RNA polymerase III (Pol III) promoters, such as 7SK, U6, and H1, are widely used for the expression of small noncoding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a noncoding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter, we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly used H1 promoter in terms of not only activity and small promoter size but also concerning safety by exclusive expression of the desired therapeutic transcript (either pol II or pol III but not both).

Optimization of a p-Coumaric Acid Biosensor System for Versatile Dynamic Performance.

Metabolic engineering is a promising approach for the synthesis of valuable compounds. Transcriptional factor-based biosensors are efficient tools to regulate the metabolic pathway dynamically. Here, we engineered the p-coumaric acid responsive regulator PadR from Bacillus subtilis. We found that yveF and yveG, two previously uncharacterized components in the sensor system, showed positive impacts on the regulation of PadR-PpadC sensor system, mostly on assisting the release of the repression by PadR. By site directed PadR engineering, we obtained two mutants, K64A and H38A, which exhibited increased dynamic range and superior sensitivity. To increase the promoter strength of the sensor system and investigate whether the PadR binding boxes can function in a "plug-and-play" manner, a series of hybrid promoters were constructed. Four of them, P1, P2, P7, and P9, showed increased strength compared to PpadC and can be regulated by PadR and p-coumaric acid. The PadR variants and hybrid promoters obtained in this paper would expand the applicability of this sensor system in future metabolic engineering research.

Construction and characterization of a zinc-inducible gene expression vector in fission yeast.

Gene expression vectors are useful and important tools that are commonly used in a variety of experiments, including expression of foreign genes, functional analysis of genes of interest and complementation experiments. In this study, a hybrid promoter, combining the adh1+ upstream activating sequence (UAS) of fission yeast and the GAL10 core promoter of budding yeast, was constructed to enable high level expression depending on the presence of zinc in culture medium for fission yeast. When the hybrid promoter was cloned on the multicopy plasmid, it was fully induced and repressed within 10 h in the presence and absence of zinc, respectively. The kinetics of induction and reduction were similar to those of the endogenous adh1+ mRNA. In contrast, native adh1+ promoter lost its tight repression in zinc-depleted condition when it was cloned on the plasmid. Because adh1+ UAS-specific transcription factors have not yet been identified, we identified UAS elements involved in zinc sensing by characterizing this hybrid promoter. We also found that the expression level increased by the TATA box mutation, GATAA, in the presence of zinc.

Cloning and transcriptional analysis of RuBisCO hybrid promoter

Cloning and transcriptional analysis of RuBisCO hybrid promoter

Tau hyperphosphorylation in the retinal ganglion cells is attenuated upon silencing of SHP2 phosphatase

AbstractBackgroundHuman and animal studies demonstrate that both amyloid precursor protein and phosphorylated Tau which are hallmark proteins associated with Alzheimer’s disease (AD) pathology are perturbed in the retinal tissues. The accumulation of pTau in the retina in addition to the brain tissue suggests eye as a valuable structure for disease diagnosis and to study the disease mechanisms in neurodegenerative disease. Recent studies in human tissues and animal models have established a molecular overlap of AD with glaucoma in the eyes and both the diseases demonstrate altered amyloid beta and Tau phosphorylation profile. We show that Shp2 interacts with Tau protein in retina and silencing this phosphatase in the retinal ganglion cells using AAV2 mediated gene therapy can protect the retina.MethodAnimal model of ocular hypertension was generated by repeat administration of polystyrene beads into the anterior chamber of rat eyes for 8 weeks resulting in sustained elevation of intraocular pressure (20.84±1.65 mmHg). Tau expression changes were investigated both in the animal model and human tissues obtained from the Sydney Eye bank (n=3) using immunohistochemical and WB. Silencing of Shp2 expression in the inner retina was achieved using intravitreal administration of AAV2 constructs under the control of CAG hybrid promoter. The animals were monitored for 2 months after Shp2 silencing and effects on Tau protein expression studied (n=48).ResultIncreased Tau Ser404 phosphorylation was observed in both human glaucoma retinas (p<0.02; student t‐test) and in the animal model of glaucoma (p<0.05) in the WB and immunohistochemical analysis. Shp2 silencing (82±4%) was achieved using AAV2 administration and transduction was monitored using GFP staining. A 7.5±3‐fold downregulation Shp2 expression was achieved using shRNAmir that resulted in significant reduction of pTau Ser404 levels in the inner retina (p<0.028, Bornferroni post‐hoc test) indicating a regulatory effect of the phosphatase on the pTau levels.ConclusionThis study demonstrates that TauSer404 phosphorylation in the retina in vivo is regulated by Shp2 phosphatase. Targeted gene therapy approaches to modulate Shp2 expression can be used as a novel strategy to reduce Tau pathology in the neural tissues in various neurodegenerative disorders.

Using a Design of Experiments Approach to Inform the Design of Hybrid Synthetic Yeast Promoters.

Hybrid promoter engineering takes advantage of the modular nature of eukaryotic promoters by combining discrete promoter motifs to confer novel regulatory function. By combinatorially screeningsequence libraries for trans-acting transcriptional operators, activators, repressors and core promoter sequences, it is possible to derive constitutive or inducible promotercollections covering a broad range of expression strengths. However, combinatorial approaches to promoter design can result in highly complex, multidimensional design spaces, which can be experimentally costly to thoroughly explore in vivo. Here, we describe an in silico pipeline for the design of hybrid promoter libraries that employs a Design of Experiments (DoE) approach to reduce experimental burden and efficiently explore the promoter fitness landscape. We also describe a software pipeline to ensure that the designed promoter sequences are compatible with the YTK assembly standard.

JSRV Intragenic Enhancer Element Increases Expression from a Heterologous Promoter and Promotes High Level AAV-mediated Transgene Expression in the Lung and Liver of Mice.

Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats. A putative intragenic enhancer, termed JE, localized to the 3′ end of the JSRV env gene, has been previously described. Herein we provide further evidence that the JE functions as a transcriptional enhancer, as it was able to enhance gene expression when placed in either forward or reverse orientation when combined with a heterologous chicken beta actin promoter. We then generated novel composite promoters designed to improve transgene expression from adeno-associated virus (AAV) gene therapy vectors. A hybrid promoter consisting of the shortest JE sequence examined (JE71), the U3 region of the JSRV long terminal repeat (LTR), and the chicken beta actin promoter, demonstrated robust expression in vitro and in vivo, when in the context of AAV vectors. AAV-mediated transgene expression in vivo from the hybrid promoter was marginally lower than that observed for AAV vectors encoding the strong CAG promoter, but greatly reduced in the heart, making this promoter/enhancer combination attractive for non-cardiac applications, particularly respiratory tract or liver directed therapies. Replacement of the murine leukemia virus intron present in the original vector construct with a modified SV40 intron reduced the promoter/enhancer/intron cassette size to 719 bp, leaving an additional ~4 kb of coding capacity when packaged within an AAV vector. Taken together, we have developed a novel, compact promoter that is capable of directing high level transgene expression from AAV vectors in both the liver and lung with diminished transgene expression in the heart.

In silico characterization of synthetic promoters designed from mirabilis mosaic virus and rice tungro bacilliform virus.

CaMV35S is the most extensively used promoter for ectopic gene expression in plant system. However, multiple use of this promoter possesses several limitation i.e. homologous based gene silencing and differential suitability in monocot and dicot plants. The strength of a promoter is defined by the presence of cis-acting elements and trans acting nucleic binding factors, thus its strength can be regulated by changing the architecture of these regulatory elements. In the present study, eight hybrid promoters were designed from two parareteroviruses, rice tungro bacilliform viruses (RTBV) and mirabilis mosaic virus (MMV). The eight hybrid promoters, along with parental promoters were characterized for the presence of functional cis-elements and transcription factor binding sites (TFBS), which were predicted using bioinformatics tools such as PLACE and Matinspector. Presence of mirabilis mosaic virus modules for specific functions and over-represented modules was determined using Model inspector. A broad range of cis-elements (85), TFBS (1471) was obtained. Presence of Dehydration responsive element binding factors, Apetala 2 (AP2), WRKY, DNA binding with one finger DOF (DOFF) motifs had shown the functional relevance of these designed promoters with abiotic stress inducibility. In addition to these stress regulating TFBS, the presence of some enhancer like motifs such as P$OCSE, P$TERE, P$TODS, P$ASRC had shown the functional relevance of these promoters as a strong candidate for enhanced expression of ectopic gene.

Expanding Toolbox for Genes Expression of Yarrowia lipolytica to Include Novel Inducible, Repressible, and Hybrid Promoters.

Promoters are critical tools to precisely control gene expression for both synthetic biology and metabolic engineering. Although Yarrowia lipolytica has demonstrated many industrially relevant advantages, promoter discovery efforts on this non-conventional yeast are limited due to the challenge in finding suitable inducible and repressible promoters. Six copper-inducible promoters and five repressible promoters were isolated in this work. Especially, Cu2+-repressible promoters showed relatively high activity under non-repressing conditions compared with a constitutive promoter, but the strength could be almost fully repressed by a supplement of a low content of Cu2+. The six Cu2+-inducible promoters were engineered to improve their dynamic regulation range with a tandem upstream activation sequence. An engineered promoter was successfully used to construct a more productive pathway for production of a novel bioproduct, wax ester, than that used for both Cu2+-inducible promoter and constitutive promoter. This study provides effective tools applicable to fine-tune the gene expression in this microbial host.

High-yield expression of extracellular lipase from Yarrowia lipolytica and its interactions with lipopeptide biosurfactants: A biophysical approach

The unconventional yeast Yarrowia lipolytica is known as a producer of extracellular lipases. Here we overexpressed extracellular lipase (YlLip2) in yeast strain Y. lipolytica AJD ΔXΔA-Lip2 harboring the overexpression cassette of the YALI0A20350 gene under the strong hybrid promoter UAS1B16-TEF. To maintain a high level of YlLip2 production, two extracellular proteases of Y. lipolytica, AEPp and AXPp, were deleted. The purified recombinant YlLip2 presented optimal catalytic activities at 37 °C and pH 8.0. The effect of two lipopeptide biosurfactants, i.e., amphisin produced by Pseudomonas fluorescens DSS73 and viscosinamide secreted by P. fluorescens DR54, on the conformation and activity of YlLip2 was evaluated using spectral methods, surface tension, and the enzyme activity assay. YlLip2 demonstrated high tolerance of the tested biosurfactants and had greater activity retention after incubation with both biosurfactants. Finally, we observed that intrinsic fluorescence intensity of YlLip2 decreased significantly with increasing lipopeptides concentration ranging from 2.5 to 60 μM. Our results showed that both biosurfactants improve enzymatic activity of YlLip2 and might suggest better interaction of the substrate with the active site. These favorable characteristics make YlLip2 a prospective additive in the pharmaceutical, food, cosmetic, and detergent industries.

Engineered dynamic distribution of malonyl-CoA flux for improving polyketide biosynthesis in Komagataella phaffii

Malonyl-CoA is a basic but limited precursor for the biosynthesis of various bioactive compounds and life-supporting fatty acids in cells. This study develops a biosynthetic system to dynamically redirect malonyl-CoA flux and improve production of malonyl-CoA derived polyketide (6-MSA) in Komagataella phaffii. A synthetic regulatory protein fusing a yeast activator Prm1 with a bacterial repressor FapR was proved to work with a hybrid promoter (-7)fapO-cPAOX1 and activate gene expression. Expression mode by the Prm1-FapR/(-7)fapO-cPAOX1 device was not affected by intracellular malonyl-CoA levels. Further, 9 promoter variants of PGAP with insertion of fapO at various sites were tested with the Prm1-FapR. It generated a biosensor of Prm1-FapR/PGAP-(+2)fapO with regulation behavior of malonyl-CoA-low-level repression/high-level derepression. Both devices were subsequently integrated into a single cell, for which fatty acid synthesis module was driven by Prm1-FapR/PGAP-(+2)fapO but 6-MSA synthesis module was expressed by Prm1-FapR/(-7)fapO-cPAOX1. The integrated system allowed continuous polyketide synthesis but malonyl-CoA-high-level “on”/low-level “off” fatty acid synthesis. This design finally increased 6-MSA production capacity by 260 %, proving the positive effects of dynamic malonyl-CoA distribution to the target compounds. It provides a new strategy for synthesis of malonyl-CoA derived compounds in eukaryotic chassis hosts.

Coupling metabolic addiction with negative autoregulation to improve strain stability and pathway yield

Metabolic addiction, an organism that is metabolically addicted with a compound to maintain its growth fitness, is an underexplored area in metabolic engineering. Microbes with heavily engineered pathways or genetic circuits tend to experience metabolic burden leading to degenerated or abortive production phenotype during long-term cultivation or scale-up. A promising solution to combat metabolic instability is to tie up the end-product with an intermediary metabolite that is essential to the growth of the producing host. Here we present a simple strategy to improve both metabolic stability and pathway yield by coupling chemical addiction with negative autoregulatory genetic circuits. Naringenin and lipids compete for the same precursor malonyl-CoA with inversed pathway yield in oleaginous yeast. Negative autoregulation of the lipogenic pathways, enabled by CRISPRi and fatty acid-inducible promoters, repartitions malonyl-CoA to favor flavonoid synthesis and increased naringenin production by 74.8%. With flavonoid-sensing transcriptional activator FdeR and yeast hybrid promoters to control leucine synthesis and cell grwoth fitness, this amino acid feedforward metabolic circuit confers a flavonoid addiction phenotype that selectively enrich the naringenin-producing pupulation in the leucine auxotrophic yeast. The engineered yeast persisted 90.9% of naringenin titer up to 324 generations. Cells without flavonoid addiction regained growth fitness but lost 94.5% of the naringenin titer after cell passage beyond 300 generations. Metabolic addiction and negative autoregulation may be generalized as basic tools to eliminate metabolic heterogeneity, improve strain stability and pathway yield in long-term and large-scale bioproduction.

Fermentation process for producing CFAs using Yarrowia lipolytica.

Past research has sought to improve the production of cyclopropane fatty acids by the oleaginous yeast Yarrowia lipolytica by heterologously expressing the E. coli fatty acid synthase gene and improving cultivation processes. Cyclopropane fatty acids display properties that hold promise for biofuel applications. The E. coli fatty acid synthase gene was introduced into several genetic backgrounds of the yeast Y. lipolytica to optimize lipid synthesis; the mean cyclopropane fatty acid productivity was 43mg L-1 h-1 on glucose, and the production rate reached its maximum (3.06g L-1) after 72h of cultivation in a bioreactor. The best strain (JMY6851) overexpressed simultaneously the E. coli cyclopropane fatty acid synthase gene under a hybrid promoter (hp8d) and Y. lipolytica LRO1 gene. In fed-batch process using crude glycerol as carbon source, JMY6851 strain displayed high lipid accumulation (78% of dry cell weight) and high biomass production (56g L-1). After 165h of cultivation, cyclopropane fatty acids represented 22% of the lipids produced; cyclopropane fatty acid productivity (103.3mg L-1 h-1) was maximal at 72.5h of cultivation.

ISEcp1-Mediated Transposition Leads to Fosfomycin and Broad-Spectrum Cephalosporin Resistance in Klebsiella pneumoniae.

A fosfomycin-resistant and carbapenemase (OXA-48)-producing Klebsiella pneumoniae isolate was recovered, and whole-genome sequencing revealed ISEcp1-blaCTX-M-14b tandemly inserted upstream of the chromosomally encoded lysR-fosA locus. Quantitative evaluation of the expression of lysR and fosA genes showed that this insertion brought a strong hybrid promoter leading to overexpression of the fosA gene, resulting in fosfomycin resistance. This work showed the concomitant acquisition of resistance to broad-spectrum cephalosporins and fosfomycin due to a single genetic event.

Engineering Prokaryotic Transcriptional Activator XylR as a Xylose-Inducible Biosensor for Transcription Activation in Yeast.

Biosensors regulated by specific substrates are needed to develop genetic tools to meet the needs of engineering microbial cell factories. Here, a xylose-inducible biosensor (xylbiosensor), comprising the Escherichia coli activation factor XylR, fusion activation domain (AD) VPRH, and a hybrid promoter with operator xylO, was established in Yarrowia lipolytica. The addition of xylose to an engineered Y. lipolytica strain harboring the xylbiosensor could trigger significant transcriptional activation of target genes, such as mcherry and the xylose utilization gene. Furthermore, a novel promoter Pleu-Pxo-Ptef was developed to construct a bidirectional expression system. The xylbiosensor showed good portability in Saccharomyces cerevisiae, suggesting its potential value in other eukaryotic cells. This study is the first to construct a "turn-on" xylbiosensor induced by xylose addition based on a prokaryotic activator XylR and eukaryotic universal AD. The xylbiosensor exhibits potential in pathway engineering for xylose utilization and xylose-derived product biosynthesis in yeast.

A Synthetic Malonyl-CoA Metabolic Oscillator in Komagataella phaffii.

Malonyl-CoA is a key metabolic molecule that participates in a diverse range of physiological responses and can act as a building block for a variety of value-added pharmaceuticals and chemicals. The cytosolic malonyl-CoA concentration is usually very low, and thus dynamic metabolic control of malonyl-CoA variation will aid its stable formation and efficient consumption. We developed a synthetic malonyl-CoA metabolic oscillator in yeast. A synthetic regulatory protein, Prm1-FapR, was constructed by fusing a yeast transcriptional activator, Prm1, with a bacterial malonyl-CoA-sensitive transcription repressor, FapR. Two oppositely regulated biosensors were then engineered. A total of 18 hybrid promoter variants were designed, each carrying the operator sequence (fapO) of FapR and the core promoter of PAOX1 (cPAOX1), which is naturally regulated by Prm1. The promoter activities of these variants, regulated by Prm1-FapR, were tested. Through this process, a sensor for Prm1-FapR/(-52)fapO-PAOX1 carrying an activation/deactivation regulation module was built. Meanwhile, 24 promoter variants of PGAP with fapO inserted were designed and tested using the fusion regulator, giving a sensor for Prm1-FapR/PGAP-(+22) fapO that contained a repression/derepression regulation module. Both sensors were subsequently integrated into a single cell, which exhibited correct metabolic switching of eGFP and mCherry reporters following manipulation of cytosolic malonyl-CoA levels. The Prm1-FapR/(-52)fapO-PAOX1 and the Prm1-FapR/PGAP-(+22)fapO were also used to control the malonyl-CoA source and sink pathways, respectively, for the synthesis of 6-methylsalicylic acid. This finally led to an oscillatory metabolic mode of cytosolic malonyl-CoA. Such a metabolator is useful in exploring potential industrial and biomedical applications not limited by natural cellular behavior.

AAV Capsid-Promoter Interactions Determine CNS Cell-Selective Gene Expression InVivo.

Cell-selective gene expression comprises a critical element of many adeno-associated virus (AAV) vector-based gene therapies, and to date achieving this goal has focused on AAV capsid engineering, cell-specific promoters, or cell-specific enhancers. Recently, we discovered that the capsid of AAV9 exerts a differential influence on constitutive promoters of sufficient magnitude to alter cell type gene expression in the rat CNS. For AAV9 vectors chicken β-actin (CBA) promoter-driven gene expression exhibited a dominant neuronal gene expression in the rat striatum. Surprisingly, for otherwise identical AAV9 vectors, the truncated CBA hybrid (CBh) promoter shifted gene expression toward striatal oligodendrocytes. In contrast, AAV2 vector gene expression was restricted to striatal neurons, regardless of the constitutive promoter used. Furthermore, a six-glutamate residue insertion immediately after the VP2 start residue shifted CBA-driven cellular gene expression from neurons to oligodendrocytes. Conversely, a six-alanine insertion in the same AAV9 capsid region reversed the CBh-mediated oligodendrocyte expression back to neurons without changing AAV9 capsid access to oligodendrocytes. Given the preponderance of AAV9 in ongoing clinical trials and AAV capsid engineering, this AAV9 capsid-promoter interaction reveals apreviously unknown novel contribution to cell-selective AAV-mediated gene expression in the CNS.