AAV Vector  Design and Construction

AAV vectors have been widely used for gene delivery and gene therapy. The first step to your success for using AAV vectors is to design a shuttle plasmid that can be efficiently packaged into AAV capsid and achieve high transgene expression. You need carefully think about the transgene expression strategies, Tag methods, AAV packaging capacity and AAV serotypes. 

AAV cloning services

At AAVnerGene, we provide One-Stop Services of Design, Construction and Cloning of Customized AAV Vectors. Send your service requirements to customer@aavnergene.com to request a quote. Our molecular and AAV experts will take care of your projects and perform the following steps:

  • ​Consultation: Comprehensive support in the selection of an appropriate cloning strategy, expression elements and AAV serotypes.

    • Choose the promoters, introns, enhancers and other regulatory elements.

    • Choose the expression and labeling strategies. 

    • Choose the AAV vectors and serotypes.

  • Preparation of DNA fragments: ​​

    • ​Amplification of requested sequences. 

    • Digestion of fragments from plasmids.

    • Synthesis of any size of DNA fragments with low cost.

  • Molecular cloning​​

    • ​Cloning of full length cDNAs, synthetic genes, PCR products or vector inserts using standard cloning techniques or Gibson assembly. 

    • Multiple fragments can be assembled together at once.​

  • Quality control: Each construct undergoes strict quality control.  

    • Restriction enzyme digestion to identify correct clones.

    • SmaI digestion to confirm two ITRs.

    • DNA sequencing to verify inserts. 

  • Delivery and documentation.The experimental strategy, raw data and final results are summarized in a detailed report.

    • ​The plasmid DNA (mini scale preparation) is delivered to customer.

    • Higher scales are available on request.

 
 
Price and time

1. Customer needs to provide templates for subclone. 

2. DNA synthesis of fragments will be charged additionally, 0.2$/bp. 

3. For complicated clones, the price may be changed.

 
Consideration 1: Expression Strategies
 

In recent years gene therapy has begun to make the advance from proof of concept to proof of practice. How to safely and efficiently deliver transgenes to target tissue in vivo is very critical for the use of gene products in clinical. Most gene therapy protocols have utilised vectors that retain their wild-type tropism (if any) and employ simple transgene expression cassettes driven by strong viral promoters. Such generic vectors have been popular because of their broad utility, and have typically proved extremely powerful in vitro and in some pre-clinical models in vivo. More recently it has become clear that modification of vector tropism to either expand or restrict gene delivery to specific tissues in vivo, and of vector expression cassettes to add further tissue selectivity, may yield significant advantages in gene therapy and serve to minimise the input titer required to evoke a phenotypic response in vivo.

The basic components of an expression cassette include promoter/enhancer elements, introns, the gene(s) of interest, and an appropriate mRNA stabilizing polyadenylation signal. Other frequently employed cis-acting elements include internal ribosome entry site (IRES) sequences to allow expression of two or more genes without the need for an additional promoter, and post-transcriptional regulatory elements(such as WPRE) to improve transgene expression.

Promoters

The sequence of the promoter region controls the binding of the RNA polymerase and transcription factors, therefore promoters play a large role in determining where and when your gene of interest will be expressed. Thus, promoter selection has received greatest attention in the literature.

Usually, the promoter region is immediately upstream of the coding sequence. This region can be short (only a few nucleotides in length) or quite long (hundreds of nucleotides long). The longer the promoter, the more available space for proteins to bind. This also adds more control to the transcription process. The length of the promoter is gene-specific and can differ dramatically between genes. Consequently, the level of control of gene expression can also differ quite dramatically between genes. 

A major limitation of AAV as a gene therapy vector is the restricted ~5 kb packaging capacity. This has largely prevented the use of large regulatory elements to control transgene expression with AAV vector systems. Customers should pay attention to this limitation and determine which expression strategies can be used.

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Models of gene therapy generally employ expression cassettes containing strong viral promoters that are constitutively active in a wide spectrum of cells. Such generic vectors have been popular because of their broad utility, and have typically proved extremely powerful in vitro and in some pre-clinical models in vivo. Common eukaryotic promoters used in research are listed below. It should be a good place to start when trying to pick your  promoter.

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More recently, it has become clear that modification of vector tropism to either expand or restrict gene delivery to specific tissues in vivo (transductional targeting), and of vector expression cassettes to add further tissue selectivity (transcriptional targeting), may yield significant advantages in gene therapy and serve to minimise the input titer required to evoke a phenotypic response in vivo. 

Endogenous eukaryotic promoters have typically proved inferior to viral promoters in terms of expression intensity, but it is likely that this may be overcome by improvements to current eukaryotic promoter constructs. Eukaryotic genes are highly regulated and it is to be expected that success in their use will depend upon the use of an appropriate promoter and the incorporation of additional elements to maximise its expression, in contrast to the frequently more compact, self-contained viral promoters. 

Tissue specific promoters provide the advantage of limiting the expression to the desired cell or tissue. However, low levels of expression and/or large size may limit their use. Moreover, each tissue/cell has many potential specific promoters, and each promoter contains different regulators. It is extremely difficult, but very important to select the best promoters to fit the size of AAV vectors. At AAVnerGene, we provides a method by combining the DNA barcoding techology with AAV vectors,  which can efficiently compare hundreds of promoters in one experiment. 

Enhancers

Enhancers are regulatory elements that activate promoter transcription over large distances and independently of orientation. While both promoters and enhancers are known to bind transcription factors , only promoters were thought to initiate transcription by RNA polymerase II (Pol II). An enhancer is a short (50–1500 bp) region of DNA that can be bound by proteins (activators) to increase the likelihood that transcription of a particular gene will occur. The enhancers, are not necessarily close to the genes they enhance. They can be located upstream of a gene, within the coding region of the gene, downstream of a gene, or may be thousands of nucleotides away.

 

Introns
 

Introns can increase gene expression without functioning as a binding site for transcription factors. This phenomenon was termed 'intron-mediated enhancement'. Introns can increase transcript levels by affecting the rate of transcription, nuclear export, and transcript stability. Moreover, introns can also increase the efficiency of mRNA translation. 

Terminators
 

The role of the terminator, a sequence-based element, is to define the end of a transcriptional unit (such as a gene) and initiate the process of releasing the newly synthesized RNA from the transcription machinery. Terminators are found downstream of the gene to be transcribed, and typically occur directly after any 3’ regulatory elements, such as the polyadenylation or poly(A) signal. The polyadenylation of a transcript is critical for nuclear export, translation, and mRNA stability. Therefore, the efficiency of transcript polyadenylation is important for transgene expression. Mammalian expression plasmids are primarily used to create mRNA and the commonly used mammalian terminators (SV40, hGH, BGH, and rbGlob) include the sequence motif AAUAAA which promotes both polyadenylation and termination. Out of those listed, the SV40 late polyA and rbGlob polyA are thought to be more efficient in terminating transcription due to the presence of additional helper sequences.In vitro studies using mammalian cultured cells have been useful in determining the effects of different polyA signals to boost expression. One study, in human epithelial-like cells, found that a transgene had a 2.5-fold increase in expression with either SV40 late or bovine growth hormone polyA (bGHpA) signal sequences compared to a minimal synthetic polyA (SPA) signal. Some of the same polyA signals were assessed in neuronal cell cultures and gave similar results; the late SV40 polyA signal and bGHpA were approximately equivalent and twice as strong as the minimal SPA. In vivo, the bGHpA signal, when packaged into AAV2 and injected intravenously into mice, gave 2- to 3-fold more transgene expression over the mouse β-globin polyA signal. Together these results suggest that polyA signal strength is independent of cell type and that in vitro results generally correlate with in vivo observations.

 
Consideration 2: Labeling Strategies

AAVnerGene has a large number of fusion tag options for customers to choose. Designing a tagging strategy requires consideration of many factors, depending on the particular application and the goals of the experiment. Send your service requirements to customer@aavnergene.com, our molecular and AAV experts will help you figure out the best strategies for your projects.

Adding tags in-frame to N-terminal or C-terminal of a native protein is a well-established strategy for many applications, including protein purification, IP, WB, and in vivo imaging.  Fluorescent proteins, such as GFP,  mCherry, tdTomato, are most often used for live cell imaging. Fluorescent protein tags enable viewing of living cells under a fluorescent microscope, or separation of live cells via FACS in real time and without any introduction of substrates. Fluorescent protein gene can be directly fused to target genes or linked with 2A self-cleaving peptides (such as T2A and P2A). Moreover, Fluorescent protein can also be introduced by IRES or even by another promoter. Small tags, such as GST, Flag, HA, C-Myc, 6His and Halo, are commonly used for protein purification, WB and IP. As the relatively small packaging capacity of AAV vectors, customer should pay additional attention to those big tags during experiments.

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Consideration 3: ssAAV, scAAV or dual AAV vectors
Single Strand AAV vectors(ssAAV)
 

The main point of consideration in the rational design of an AAV vector is the packaging size of the expression cassette that will be placed between the two ITRs. Regular single strand AAV(ssAAV) has a packaging capacity of ~5.0 Kb. Thus, as a starting point, it is generally accepted that anything under 5 kb (including ITRs) is sufficient. Since the two ITRs of AAV are about 0.2-0.3Kb total, the foreign sequence between two ITRs should be smaller than 4.7 Kb. When the length of inserted DNA  is close to the maximum, the packaging efficiency decreases significantly. If the insert DNA is over 4.7 Kb, only partial of DNA is packaged into AAV vectors. For large coding sequences, the use of dual, overlapping vector strategies may be a good  choice for you.

 
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Self complementary AAV vectors(scAAV)

scAAV is a viral vector engineered from the naturally occurring AAV to be used as a tool for gene therapy. This lab-made progeny of AAV is termed "self-complementary" because the coding region has been designed to form an intra-molecular double-stranded DNA template. A rate-limiting step for the standard AAV genome involves the second-strand synthesis since the typical AAV genome is a single-stranded DNA template. However, this is not the case for scAAV genomes. Upon infection, rather than waiting for cell mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and  transcription. The caveat of this construct is that instead of the full coding capacity found in rAAV (5.0 kb).  scAAV can only hold about half of that amount (≈2.5kb).

 
Dual and overlapping AAV vectors
Oversize packaging
 

Researchers have shown successful expression of 9 kb gene following transduction with AAV vectors in which they had attempted to package oversize transgenes. This success was elucidated to have resulted from the packaging of fragmented transgenes. When a transgene is large, packaging that begins from the 3’ ITRs of both the plus and minus strands and becomes truncated at an undefined point, therefore each capsid carries an incomplete fragment of transgene. This results in a mixed population of AAV vectors carrying different truncated lengths of the transgene plus and minus strands. The successful generation of target product despite this heterogeneous vector population was deduced to result from the plus and minus strands carrying overlapping regions of the original therapeutic transgene that could undergo homologous recombination (HR) or annealing at the complementary regions prior to second-synthesis.


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Dual functional vectors

Some large genes can be separated into two functional fragments, such as FVIII.  For those genes,  one AAV vector is used to carry the N terminal with an addtional stop codon. Another AAV vector is used exclusively to express the C terminal with additional start codon. The two AAV vectors can be produced separately. The combination of the two vectors can generate a functional full protein .

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Overlapping AAV Dual Vectors

An advancement on the fragmented dual vector approach is the overlapping approach. In this strategy, there are two defined transgenes that each carry a demarcated fragment of the therapeutic gene CDS that includes a portion of specified sequence overlap in each transgene. The overlapping strategy relies on the same premise that enables the fragmented approach, whereby a region of sequence overlap initiates joining of two separate fragments into a single larger one.

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Trans-splicing AAV Dual Vectors

This strategy has no region of sequence overlap and therefore the two transgenes are completely distinct and contain two different fragments of the therapeutic gene. The approach relies on the tendency of ITRs to concatemerize as it has been shown that following transduction and second-strand synthesis, AAV transgenes form stable episomal structures through joining of their ITR structures, a process known as concatemerization. The trans-splicing approach piggy-backs on this process and so with appropriate dual vector design, following joining of the ITRs from the dual vectors, the concatemerized ITR structure that would lie in the middle of the therapeutic gene can be removed by native cellular mechanisms during transcription due to the inclusion of a splice donor site following the 3’ end of the gene contained in the upstream transgene and a splice acceptor site prior to the 5’ end of the gene contained in the downstream vector.

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Trans-splicing and overlapping AAV Dual Vectors

With the trans-splicing approach, there is a concern that the dual vector transgenes will join in an undesirable way or not concatemerize at all. With the overlapping approach, a concern is that concatemerization would occur at all as there would be no feature to remove an unwanted ITR structure present in the middle of a CDS. The hybrid strategy counters both these concerns by combining the two approaches. This hybrid dual vector strategy incorporates both an overlap region and splice donor/splice acceptor sites in the dual vector transgenes.

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Consideration 4: AAV serotypes

To date, a number of AAV serotypes and over 100 AAV variants have been isolated from adenovirus stocks or from human/non-human primate and other animal tissues. Utilization of alternative AAV serotypes can not only lower the vector load due to their potentially higher transduction efficiency, but also help evade preexisting neutralizing antibodies generated as a result of humoral immune response to natural infection or prior treatment with AAV-based vectors. In addition, AAV serotypes and variants can serve as templates for design of tissue-targeted capsid constructs that will serve to expand and complement the current range of AAV vectors. Thus, it is very important to figure out naturally occurring AAV serotypes and engineered hybrid AAV vectors that can achieve high level of transduction in specific tissue types. 

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Currently, the common way to compare AAV variants is to test them side-by-side individually, whereas it is difficult to generate, control and handle hundreds of AAV vectors in every lab or company while limited candidates decrease the chance to find better AAV variants.

AAVnerGene's ATHENA I AAV serotpye selection platform, which allows us to fastly and semi-high-throughputly compare the AAV variants both in vivo and in vitro.​​