She has been extremely kind and supportive as a friend and as a mentor during the most difficult times in my personal life. Aarathi Shanmugam, my friend since my undergraduate years, has been with me every step of the way ever since and am very lucky to have her in my life.
PUBLISHED CONTENT AND CONTRIBUTIONS
Multiply-spawn selection yields AAV vectors targeted to different cell types of the central nervous system after systemic delivery. Develop AAV vectors for more efficient and selective gene expression in specific cell types of the nervous system after systemic delivery.
BACKGROUND AND MOTIVATION
- VECTORS FOR GENE DELIVERY
- ADENO-ASSOCIATED VIRAL VECTORS FOR GENE DELIVERY
- ENGINEERING AAV CARGOS FOR EFFICIENT AND TARGETED PROTEIN EXPRESSION EXPRESSION
- ENGINEERING AAV CAPSIDS TO MODIFY THE VECTOR TROPISM
- IN VIVO SELECTION OF AAV CAPSIDS USING CREATE
All of the above characteristics make them a safer choice as a gene delivery vector than other viruses. Stop codons inserted into the cap gene eliminate the expression of VP1, VP2, and VP3 capsid protein. e) DNase-resistant AAV vector genomes (vg) produced with the split AAV2/9 Rep-AAP and rAAV-Cap-in-cis-lox genome (top) compared to the vg produced with standard AAV2/9 Rep-Cap -helper and rAAV-UBC-mCherry genome (middle) or with the AAV2/9 Rep-AAP and rAAV-UBC-mCherry genome (bottom). f) Cloning the 7-mer capsid library into the rAAV-ΔCap-in-cis vector. g) The AAV9 surface model shows the location of the 7-mer inserted between amino acids (aa) 588 and 589 (magenta).
CHARACTERIZATION AND PRODUCTION OF SYSTEMIC AAV VECTORS FOR WIDESPREAD AND TARGETED GENE
DELIVERY IN RODENTS
SUMMARY
INTRODUCTION
Interestingly, AAV-PHP.B shows low transduction in the brain when administered systemically to BALB/cJ164 mice (Figure 2.5a). AAV-PHP.B transduces the brain more efficiently than AAV9 in FVB/NCrl and 129S1/SvImJ mice (Figure 2.5a).
PROCEDURE
Critical Step: The transfection solution will appear slightly cloudy due to the formation of DNA-PEI complexes178,179. Critical Step: The pellet may be soft, making it difficult to extract all the supernatant. Use a P200 pipette to transfer the virus directly from the upper chamber of the Amicon filter device to a 1.6 ml screw cap vial; store at 4°C.
Use a P200 pipette to transfer the virus from the upper chamber of the Amicon filter device directly to a Costar Spin-X filter unit inside a centrifuge tube. Critical step: When dipping the tip of the pipette into the virus stock, insert the tip just below the surface of the liquid rather than plunging deep into it. The needle should be placed behind the eyeball in the retro-orbital sinus.
TROUBLESHOOTING
Use a forward twisting motion to insert the needle into the tube (Supplementary Video Practice on an OptiSeal tube filled with water. Use the tube removal tool to remove the black cap from the tube after inserting the needle but before collecting the virus (Fig. 8g and supplementary video practice in an OptiSeal tube filled with water Density gradient flows from the needle hole into the tube after the needle is removed.
For further viral preparations, ensure that the black cap is replaced before removing the needle from the tube (supplementary video of an exercise on an OptiSeal tube filled with water. Load more virus into the same insulin syringe, place the eyeball needle into the retro-orbital sinus and try by injecting again Place the needle for the eyeball in the retro-orbital sinus, never puncture the eye itself.
TIMING
Users can measure production efficiency for each experiment by running a positive control (e.g. pAAV-CAG-eYFP). The chosen method for evaluating transgene expression will vary from user to user and may include molecular, histological and/or functional approaches220 (step 50). We typically use fluorescent reporters to assess gene expression in thick (≥100 µm), cleared tissue samples; below we discuss the expected results for the applications presented here (Figures 2.2–2.5) and in our previous work133,157.
Most markers, including mTurquoise2, mNeonGreen and mRuby2, can also be detected after mounting the labeled tissues in optical clearing reagents such as RIMS218 or ScaleSQ170 (Figures 2.3a,c,d, 2.4b-d and 2.5e available in media (mounting in the market) 2.5a,c). Depending on the rAAV genome, fluorescent proteins can localize to specific cellular compartments, including the nucleus (via NLS) (Figure 2.2a,b), cytosol (Figures 2.2d,e, 2.3, 2.4b and 2.5) and cell membrane (via farnesylation175 or fusion with a membrane protein such as ChR2) (Figures 2.3d and 2.4d). In summary, we present a comprehensive protocol for the production and administration of AAV-PHP viruses.
SUPPLEMENTARY INFORMATION
We validated the ability of AAV-PHP variants to provide efficient and non-invasive gene delivery to specific cell populations throughout the body. Supplementary Table 2.ST1: Use of AAV-PHP capsids for efficient transduction between organs and cell populations. Species/strains, organs and cell populations investigated to date following intravenous administration of AAV-PHP viruses.
This is an interactive calculator and is provided as an Excel file (see Step 2 and "Reagent Setup"). The 15% layer contains high salt to destabilize ionic interactions between viral particles and cellular proteins in the clarified lysate183. This is an interactive calculator and is provided as an Excel file (see Step 42 and "Reagent Setup").
MULTIPLEXED CRE-DEPENDENT SELECTION (M-CREATE) YIELDS SYSTEMIC AAVS FOR TARGETING DISTINCT BRAIN CELL TYPES
SUMMARY
INTRODUCTION
Previously, we developed the AAV-PHP.B and AAV-PHP.eB variants of AAV9 using a selection method called CREATE157. This method applies positive selective pressure for capsids capable of infecting a target cell population by linking a viral genome containing lox sites with in vivo selection in transgenic mice expressing Cre in the cell type of interest. Lox recombination-dependent PCR amplification of only those capsids that successfully deliver their genomes to the nuclei of the target cell type.
3.S1a,b), which compares the enrichment profiles of thousands of capsid variants in multiple cell types and organs within a single experiment. This method improves on its predecessor by capturing the breadth of capsid variants at each stage of the selection process. These improvements allow interpretation of the relative infectivity of variants over a wide range of enrichments in multiple positive selections and enable post-hoc negative screenings by comparing capsid libraries obtained from multiple target cell types or organs.
RESULTS
This amino acid pattern is very similar to the TLAPFK motif in the previously identified variant AAV-PHP.B157. AAs are colored by the shared identity of AAV-PHP.B and eB (green) or under new variants (unique color per position). Despite the sequence similarity to AAV.PHP.B, the tropism of AAV-PHP.V1 is focused on transducing brain vascular cells (Figure 3.3c and Supplementary Figure 3.S4a).
When delivered intravenously, AAV-PHP.V1 carrying a fluorescent reporter under the control of the ubiquitous CAG promoter transduces ∼60%. The enrichment of AAV-PHP.eB and AAV-PHP.N across all libraries is mapped on the plot. We tested the previously mentioned AAV-PHP.C1 (RYQGDSV), as well as AAV-PHP.C2 (WSTNAGY), and AAV-PHP.C3 (ERVGFAQ) (Figure 3.6a).
DISCUSSION
The table provides a list of primers used in M-CREATE throughout the different steps of the selection process as described in Methods. For vector production, about 10 ng of the purified and assembled library was used to transfect each 150 mm plate of 293T cells, and we obtained a yield of about 6×1011 vg per 150 mm plate (i.e., the R2 yield was six times higher than that of R1, surprising given that these sequences are already produced well enough to survive R1 selection). To extract rAAV DNA from the purified rAAV viral library, ∼10% of the purified viral library was used to extract the viral genome by proteinase K treatment.
The dose was determined based on the virus yield, which was different across rounds of selection (Supplementary Figure 3.S2a). The quality of the aligned data was further examined to remove any erroneous sequences (such as those with stop codons). For the amino acid frequency plots, the number at the bottom represents the position of the diversified motif from 1.
SUPPLEMENTARY INFORMATION
Higher magnification images of AAV-PHP.V2 transduced brain sections stained with αGLUT or αS100 or αOlig2 (magenta) are shown. b), cerebrovascular transduction with AAV-PHP.V2 carrying ssAAV:CAG-DIO-EYFP (green) in adult Tek-Cre mice is shown (left, 1x1012 vg i.v. dose per mouse, 4 weeks of expression) and efficacy ( right) is. Supplemental Figure 3.S7: Evolution of the AAV-PHP.B capsid by diversification of amino acid positions 587-597. a), shows the distribution of brain libraries R1 and (b), R2 (at AA, SS RC level, ranked in descending order of scores). The SS for AAV-PHP.N and AAV-PHP.eB in the libraries are mapped in the enlarged view of this plot (box with dotted line).
Background autofluorescence is in magenta. f), Transduction of mouse brain with the AAV-PHP.N variant carrying the CAG promoter driving the expression of mNeonGreen (n = 3, 1x1011 vg i.v. dose per C57BL/6J adult mouse, 3 weeks of expression). Supplementary Figure 3.S8: Examination of AAV-PHP variants across different mouse strains and in vitro human brain microvascular endothelial cells. a), Transduction of AAV9, AAV-PHP.eB and AAV-PHP.V1 in human brain microvascular endothelial cell culture (HBMEC) is shown. Supplementary Video 3.V1: Brain-wide transduction of endothelial cells by systemic delivery of the AAV-PHP.V1 capsid.
FUTURE DIRECTIONS
- EXPANSION OF AAV VECTOR TOOLKIT
- INVESTIGATION OF MECHANISM OF ENGINEERED VECTORS
- DEVELOPMENT OF TECHNOLOGIES TO EMPOWER M-CREATE
- POTENTIAL APPLICATIONS OF AAV VECTOR TOOLKIT
For example, AAV-PHP.B vectors that worked very well in selected rodent strains use a cell surface protein receptor, ly6a, whose homologue is not seen in humans. Therefore, studies similar to those performed to identify ly6a for translational purposes will be very useful in making an informed decision about the choice of the AAV-PHP.C vectors for translation. For example, AAV-PHP.B vectors were previously used in basic neuroscience studies in mice in neurological disease models158 and in preclinical gene therapy applications161,276,277.
And one can hope to see similar applications from vectors like AAV-PHP.N or AAV-PHP.Cs. The AAV-PHP.V variants will open up new applications in the field investigating BBB malformations widely associated with various neurodegenerative diseases, including Alzheimer's247,249, or aging studies278 or other neuropathologies215,279,280. For applications beyond rodents, further studies are required for AAV-PHP.Cs across species to determine their utility as a gene delivery vector.
BIBLIOGRAPHY
Intracellular trafficking of adeno-associated virus vectors: routing to the late endosomal compartment and proteasomal degradation. Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain. Adeno-associated virus (AAV) serotypes have characteristic interactions with domains of the cellular AAV receptor.
Rational design and engineering of a modified adeno-associated virus (AAV1)-based vector system for enhanced retrograde gene delivery. In vivo directed evolution of a novel adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous. Designer gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors for enhanced gene transfer.
