재조합 adenovirus 벡터는 다양한 포유류 세포 유형에서 높은 수준의 transgene 발현을 달성하기 위해 사용되는데, 벡터는 호스트 게놈에 통합되지 않고 episomal DNA로 남아 있다. 높은 transduction 효율과 높은 수준의 단기 유전자 발현이 adenovirus 벡터를 in vivo 유전자 전달에 선호하는 툴로 만들어, 유전자 치료와 백신에 자주 사용된다.
VectorBuilder는 특히 벡터 클로닝 서비스에 사용되는 adenovirus 벡터 시스템을 위해 titer, 순도, 생존력 및 일관성 측면에서 재조합 adenovirus 생산 프로토콜을 크게 개선한 독점 기술과 시약을 개발했다. 그 결과 adenovirus 벡터 클로닝과 adenovirus 패키징에 대한 니즈에 만족하여 당사에 반복적으로 의뢰하는 고객들이 많아지고 있다
Types of adenovirus offered
- Human Ad5 adenovirus
- Chimeric Ad5/F35 adenovirus
Price and turnaround
|Scale||Application||Typical Titer||Minimum Titer||Volume||Price (USD)||Turnaround|
|Pilot||Cell culture||>2x1010 IFU/ml||>1010 IFU/ml||250 ul (10x25 ul)||$399||20-32 days|
|Medium||1 ml (10x100 ul)||$599|
|Large||>2x1011 IFU/ml||>1011 IFU/ml||1 ml (10x100 ul)||$999|
|Ultra-purified medium||Cell culture & in vivo||>2x1012 VP/ml||>1012 VP/ml||500 ul (10x50 ul)||$1,299||22-36 days|
|Ultra-purified large||1 ml (10x100 ul)||$1,599|
IFU = Infectious units; VP = Virus particles
|For non-ultra-purified scales||For ultra-purified scales|
|Your custom adenovirus||Your custom adenovirus|
Free: control virus
Add-on purchase (optional): ultra-purified control virus
Control adenovirus 벡터는 custom 바이러스의 생물학적 응용과 일치하도록 디자인되어, adenovirus transduction을 테스트하는 데 사용된다. 예를 들어 custom 바이러스가 유전자를 과발현하는 경우, 제공되는 control virus는 EGFP control adenovirus (adenovirus overxpressing EGFP)이고, custom 바이러스가 유전자에 대해 shRNA를 발현하면 제공된 control virus는 scramble shRNA를 발현한다. Control virus에 대한 자세한 정보는 다음과 같다.
|Vector System||Control Virus Vector Name||Control Virus Vector ID|
|Adenovirus gene expression system||pAV[Exp]-CMV>EGFP||VB150925-10024|
|Adenovirus U6-based shRNA knockdown system||pAV[shRNA]-EGFP-U6>Scramble_shRNA||VB161026-1127kyf|
|Adenovirus miR30-based shRNA knockdown system||pAV[miR30]-CMV>EGFP:Scramble_miR30-shRNA||VB180124-1101mjs|
|Adenovirus CRISPR system||pAV[Exp]-CMV>EGFP||VB150925-10024|
Shipping and storage
Non-ultra-purified adenovirus는 HBSS buffer에 저장되며, ultra-purified adenovirus는 GTS buffer에 저장된다. 모든 adenovirus는 드라이아이스로 운송된다. Adenovirus는 수령 즉시, 장기보관을 위해서는 -80℃ (1년 이상 안정 가능) 또는 단기 보관을 위해서는 -20℃ (예: 2-3주)에서 보관하여야 한다. Adenovirus는 다른 많은 바이러스(예: Lentivirus) 보다 안정성이 뛰어나고 여러번의 동결과 융해에도 바이러스 활성의 손실은 적지만, 실제로는 반복적인 동결-융해 사이클은 피해야 한다.
Human Ad5 adenovirus
Chimeric Ad5/F35 adenovirus
Adenovirus production and QC
For our adenovirus manufacturing, the adenoviral vector carrying the gene of interest (GOI) is first linearized by restriction digestion with PacI. The linearized vector DNA is transfected into packaging cells expressing the adenovirus gene E1 to produce recombinant adenovirus, and adenoviral particles are released into tissue culture medium where they are collected. For ultra-purified adenovirus (in vivo grade), viral particles are further purified and concentrated by cesium chloride (CsCl) gradient ultracentrifugation. We measure the titer using an immunocytochemistry-based approach to detect the adenovirus-specific hexon protein. For ultra-purified adenovirus, we measure the optical density (using OD260) of the viral particles to estimate the titer.
For each adenovirus produced by VectorBuilder, quality control includes titer measurement, sterility testing for bacteria and fungi, and mycoplasma detection. If the adenoviral vector encodes a fluorescent protein, we would perform transduction test to detect corresponding fluorescence. Additionally, for ultra-purified adenovirus, we routinely perform endotoxin assay to check the endotoxin level.
Chimeric Ad5/F35 adenovirus production and QC
For our chimeric Ad5/F35 adenovirus manufacturing (Figure 1), the Ad5/F35 adenoviral vector carrying the gene of interest (GOI) is first linearized by restriction digestion with PacI. The linearized vector DNA is transfected into packaging cells expressing the adenovirus gene E1 to produce recombinant adenovirus, and adenoviral particles are released into tissue culture medium where they are collected. For ultra-purified Ad5/F35 adenovirus (in vivo grade), viral particles are further purified and concentrated by cesium chloride (CsCl) gradient ultracentrifugation. We measure the titer using an immunocytochemistry-based approach to detect the adenovirus-specific hexon protein. For ultra-purified Ad5/F35 adenovirus, we measure the optical density (using OD260) of the viral particles to estimate the titer.
Figure 1. Typical workflow of chimeric Ad5/F35 adenovirus packaging.
For each adenovirus produced by VectorBuilder, quality control includes titer measurement, sterility testing for bacteria and fungi, and mycoplasma detection. If the Ad5/F35 adenoviral vector encodes a fluorescent protein, we would perform transduction test to detect corresponding fluorescence. Additionally, for ultra-purified adenovirus, we routinely perform endotoxin assay to check the endotoxin level.
Ad5 adenovirus vs. chimeric Ad5/F35 adenovirus
While human adenovirus serotype 5 (Ad5) vectors are most widely used for adenovirus-based applications, a major limitation associated with such vectors is their dependence on the coxsackie and adenovirus receptor (CAR) for infecting target cells. Host cells that completely lack or have insufficient expression of CAR cannot be efficiently transduced with Ad5 vectors. Ad5/F35 vectors help overcome this limitation by expressing a chimeric fiber protein comprised of the knob and shaft derived from adenovirus serotype 35 (Ad35) and the fiber tail derived from Ad5. This enables Ad5/F35 adenoviral vectors to readily transduce CAR-negative cells or cells with low levels of CAR-expression in addition to CAR-positive cells with high levels of CAR expression, by exploiting the ability of the Ad35 fiber protein to attach to target cells via the non-CAR receptor, CD46. The chimeric design of Ad5/F35 adenovirus has been highly instrumental in expanding the tropism of adenoviral vectors to cell lines such as hematopoietic cells, primitive stem cells, vascular smooth muscle cells and CAR-negative tumor cells which otherwise exhibit poor transduction efficiency with conventional Ad5 vectors.
We have developed a number of proprietary techniques to optimize our Ad5/F35 packaging protocol. Our optimized Ad5/F35 adenovirus has been validated to exhibit much higher transduction efficiencies in cells with insufficient CAR expression (Figure 2) than compared to conventional Ad5 adenovirus.
Figure 2. K562 cells were transduced with EGFP expressing Ad5 or Ad5/F35 adenovirus at increasing MOI and EGFP positive cells were analyzed by flow cytometry at 48 hours post-transduction. K562 cells exhibit lows levels of CAR expression as shown by previous studies.
How to Order
Order both vector construction and virus packaging
Order virus packaging of your own vector
If customer-supplied lentiviral plasmids are used in packaging, please send them to us following the Materials Submission Guidelines. Please strictly follow our guidelines to set up shipment to avoid any delay or damage of the materials. All customer-supplied materials undergo mandatory QC by VectorBuilder which may incur $100 surcharge for each item. Please note that production may not be initiated until customer-supplied materials pass QC.
What features does Adenovirus have comparing to other viral vectors?
|Tropism||Broad||Broad||Ineffective for some cells||Depending on viral serotype|
|Can infect non-dividing cells?||Yes||No||Yes||Yes|
|Stable integration or transient||Stable integration||Stable integration||Transient, episomal||Transient, episomal|
|Maximum titer||High||Moderate||Very High||High|
|Primary use||Cell culture and in vivo||Cell culture and in vivo||In vivo||In vivo|
|Immune response in vivo||Low||Low||High||Very low|
How is viral titer determined in VectorBuilder?
For adenovirus, we measure the functional titer. After transducing serially-diluted adenovirus into 293A cells, we use an immunocytochemistry-based approach to count the number of cells being successfully transduced via the detection of adenovirus-specific hexon protein, and each immunostained cell is considered as one infectious unit. Cells are infected at very low multiplicity of infection (MOI) to ensure that most transduced cells are each infected by a single viral particle. This assay shows good correlation with conventional plaque assay. For ultra-purified adenovirus, we directly measure the optical density (using OD260) of the viral particles to estimate titer, because there is a tight correlation between the optical density of ultra-purified adenovirus and functional titer. Adenovirus has very good stability. In our preparation, the viral particles are essentially all alive and can remain functional at room temperature for many days.
What is VectorBuilder's guarantee for virus titer and turnaround?
We guarantee the minimum titer when the region of the vector being packaged into virus (from 5’ ITR to 3’ ITR) is below the adenovirus cargo limit (38.7 kb). For sizes above this, the adenoviral genome may be unstable, and rearrangement may occur during packaging. Additionally, we are not able to guarantee titer for the following vectors:
- Vectors containing sequences that could adversely affect the packaging process such as toxic genes (e.g. proapoptotic genes), genes that compromise the integrity of packaging cells or virus (e.g. membrane proteins that cause cell aggregation), and sequences prone to rearrangements or secondary structures (e.g. repetitive or highly GC-rich sequences);
- Customer-supplied plasmids containing undisclosed sequences or atypical adenoviral functional elements (e.g. ITR) that may introduce uncertainties to packaging efficiency.
The estimated turnaround is the time from production initiation to completion. It does not include waiting time for any customer-supplied materials (e.g. template DNA or viral vectors), QC of such materials, and transit time for shipping final deliverables to the customer.