Efficient Gene Knockdown in the Liver via Intrasplenic Injection of Adeno-Associated Virus Serotype 8 (AAV8)-Delivered Small Hairpin RNA

Summary

This protocol describes how intrasplenic injection of AAV8-delivered small hairpin RNA achieves the same gene knockdown efficiency in the liver as portal vein injection, representing a simpler procedure with much lower perioperative and postoperative mortality and complications.

Abstract

The liver is a major organ that performs essential metabolic functions. Developing an efficient and safe method to knock down gene expression in the liver provides an important tool for determining gene function in liver pathophysiology. In this study, we describe a method for intrasplenic injection of adeno-associated virus serotype 8 (AAV8) engineered to express a small hairpin RNA (shRNA) against a target gene of interest, nucleostemin (NS). Intrasplenic injection of AAV8 expressing an NS-targeting shRNA (AAV8-shNS1) achieved the same knockdown efficiency of NS in the liver as did portal vein injection, compared to the injection of AAV8 expressing a scrambled sequence shRNA (AAV8-shScr). Furthermore, injection of the AAV8-shRNA virus triggered minimal inflammatory reactions in the liver parenchyma. Most importantly, this intrasplenic injection protocol was not technically demanding and caused minimal bleeding at the injection site, which is the leading cause of perioperative and postoperative mortality when performing portal vein injection. This study reports an improved and relatively safe method to achieve efficient gene knockdown in the liver.

Introduction

The liver is a vital organ that metabolizes nutrients and chemicals, but is also under constant exposure to cytotoxic and carcinogenic insults. While the adult liver is capable of regrowing after injury, its regenerative power is severely hampered by age¹. To date, the only therapeutic option for patients with end-stage chronic liver diseases or massive acute liver damage is liver transplantation, which poses many challenges of its own². To better understand the molecular pathophysiology of the liver by interrogating the functions of genes of interest, genetic manipulations have been developed for in vivo application, including RNAi-mediated knockdown and targeted deletion by a Cre recombinase in the presence of loxP sites³. Cre-expression cassette and shRNA construct can be delivered by viral vehicles.

Adeno-associated virus serotype 8 (AAV8) is a robust vector for gene delivery to selective tissue types (e.g., liver, skeletal muscle, heart, brain, and pancreas) with high efficiency and low inflammatory response^4,5. To target internal body organs, AAV8 is commonly introduced by tail vein injection, in which viral particles first travel through the lung before reaching the systemic circulation. In contrast, portal vein injection allows them to reach the liver first before circulating through the lung, a potential source of sequestering and dilution. However, portal vein injection is technically challenging and often complicated by operation-induced bleeding and a high mortality rate. To achieve an efficient gene knockdown (KD) in the liver while avoiding the issue of high peri/postoperative mortality, we tested a method of intrasplenic injection of AAV8 carrying an engineered shRNA targeting nucleostemin (NS) (AAV8-shNS1) and compared its KD efficiency to that of portal vein-injected AAV8-shNS1.

NS is a stem/progenitor cell-enriched protein discovered first in neural stem cells and later in several other types of stem cells and cancers^6,7. The biological importance of NS is shown by the early embryonic lethal phenotype of germline NS-knockout (NSKO) mice in vivo^3,8 and by NSKD-induced perturbation of self-renewal in vitro^9,10. In adult animals, high levels of NS expression are found in the testis and several tissues undergoing regeneration, including the dedifferentiating newt-pigmented epithelial cells after lentectomy and muscle cells after limb amputation¹¹, as well as regenerating mammalian tissues, such as mouse cardiomyocytes after cardiac injury¹² and hepatocytes after liver injury (e.g., CCl₄) or surgical resection (e.g., partial hepatectomy)^13,14. Furthermore, NS has been shown to play important roles in the development of mammary, liver, and oral tumors^15,16,17. Mechanistically, NS has been shown to promote self-renewal by protecting the replicating genome from DNA damage^18,19. However, due to the early embryonic lethal phenotype of germline NSKO, an experimental method to temporally introduce NSKD after the completion of tissue development is needed to further determine its biological activities in adult organs.

In this article, we use NS as a case in point to illustrate the establishment and testing of an in vivo liver gene KD method that is efficient and has a low procedure-induced mortality.

Protocol

All animal experiments completed in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the Texas A&M University Health Science Center in Houston (approval number: 2021-0264-IBT) and performed in accordance and compliance with all relevant regulatory and institutional guidelines. Female C57BL/6J mice (4-5 months old, body weight 23-30 g) were used. The details of the reagents and equipment used are listed in the Table of Materials.

1. AAV8- shRNA design and production

NOTE: Please refer to Sands et al.⁴ for procedural details.

1. Design sense and anti-sense shRNA oligonucleotides that target a 21-nucleotide long sequence within the coding region of the gene of interest. In this case, mouse NS (shNS1) and a scrambled sequence of 21 nucleotides as negative control (shScr) were used (Figure 1A, bottom panel).
2. Include in the hairpin structure a stem-loop sequence of TTCAAGAGA, a 5' blunt end, a 3' XhoI-compatible cohesive end, and a gene-specific targeted sense sequence of 5'-GAA CTA AAA CAG CAG CAG AAA-3' (shNS1) or a scrambled sequence of 5'-TCT CGC TTG GGC GAG AGT AAG-3' (shScr).
3. Phosphorylate and anneal each pair of oligonucleotides.
4. Digest AAV gene transfer vector, AV5-siRNA-GFP (Figure 1A, top panel), with HpaI and XhoI, dephosphorylate, and gel purify the vector fragment.
5. Ligate annealed oligonucleotides into the gel-purified vector.
6. Transform ligated DNA products into competent DH5α bacteria with subcloning efficiency.
7. Pick single clones by ampicillin resistance and grow 100 mL bacterial culture for plasmid miniprep. Confirm correct cloning by sequencing and prepare plasmids using the commercially available endotoxin-free kit (following the manufacturer's instructions).
8. Pack AAV8 viruses by transfecting the AV5 transfer vector, Rep/Cap serotype 8, and AdF6 helper plasmids into 293T cells using iMFectin transfection reagent. A total of 40 × 15 cm dishes were transfected and harvested/digested at 3-day post-transfection.
9. Recover cell-associated AAV8 by cell lysis and precipitate media-secreted AAV8 with 40% PEG. After digestion, combine AAV8 viruses in the cell lysate and the medium, purify on a discontinuous iodixanol gradient, and concentrate in molecular weight cut-off centrifugation units (1,00,000 MW).
10. Quantify AAV8 titers by absolute endpoint qPCR with primers: WPRE-172 (5'- TTTATGAGGAGTTGTGGCCC-3') and WPRE-392 (5'- CAACACCACGGAATTGTCAG-3').

2. Intrasplenic injection of AAV8 (Figure 1B, 1C) 

1. Place C57BL6/J mice in an anesthesia chamber filled with 2% isoflurane and oxygen (following institutionally approved protocols). After loss of consciousness, transfer mice to the aseptic surgical area in a right lateral recumbent position. Maintain anesthesia by 2% isoflurane with oxygen delivered via a nose cone.
2. Sterilize the incision site with 2% chlorhexidine followed by 80% ethanol. Use a pair of 6.3-inch medical scissors to make a 15 mm incision on the left upper abdominal wall, followed by a 10 mm incision on the peritoneum.
3. Gently expose the spleen using forceps and hold it in place with a wet gauze placed underneath the organ.
4. Gently mix and inject AAV8 viruses (50 μL) over a 30 s period into the spleen using a 30 G needle with the tip placed at a depth of 3 mm below the surface (Figure 1B). Cover the injection site with a piece of cotton swab and compress for 1 min.
5. Suture the peritoneum with a 4-0 absorbable chromic gut suture.
6. Close the abdominal wall and skin with a 4-0 silk suture.
7. Place mice in a clean cage on top of an electrical heat pad and monitor them until recovery.

3. Portal vein injection of AAV8

1. Anesthetize C57BL6/J mice by isoflurane inhalation (2%) mixed with oxygen (following institutionally approved protocols).
2. Perform laparotomy^4,13 through a midline incision (~1 inch) on the skin and then into the peritoneum.
3. Place a sterile gauze pad soaked in sterile saline on the left side of the mouse and use a sterile cotton swab to gently pull out the large and small intestine. Place the intestine on top of a wetted gauze and cover it with a pad to avoid contacting the skin or drying.
4. Inject the AAV virus in the portal vein with a 32 G sterile needle in a volume of 50 μL. Insert the needle 3-5 mm into the port vein at an angle less than 5 degrees. Allow blood to flow past the needle for 5 s to avoid backflow.
5. Place a sterile cotton swab tip on top of the injection site with gentle pressure until the entire closure of the vein and no bleeding (about 5 min) to avoid bleeding.
6. Gently put internal organs back into the abdominal cavity.
7. Close abdominal wall and skin with 4-0 silk suture.

4. qRT-PCR analysis

1. Extract RNAs from 50-100 mg of liver tissue^4,13 using a commercially available RNA isolation reagent following the manufacturer's instructions (see Table of Materials).
2. Synthesize 1^st strand cDNAs from total RNAs with random hexamers and M-MLV reverse transcriptase^20,21.
3. Determine the ΔC(t) values between the target gene and reference genes using the single-color real-time PCR detection system and supermix SYBR green reagent following previously published reports^22,23.
4. Measure the ΔΔC(t) values from four biological replicates and three technical repeats (n = 12). Set T_m at 60 °C for all reactions. Confirm results using two reference genes, Rps3 and Rplp0.
5. Design primer sequences as follows: Ns, 5'-gtc tga tct agt acc aaa gg-3' and 5'-ggg aaa cca atc act cca ac-3'; Rps3, 5'-atg gcg gtg cag att tcc aa-3' and 5'-cat tct gtg tcc tgg tgg c-3'; Rplp0, 5'-ctg aag tgc tcg aca tca ca-3' and 5'-agt ctc cac aga caa tgc ca-3'.

Results

Efficiencies of gene KD in the liver by intrasplenic vs. portal vein injection of AAV8-shRNA
AAV8 viral stocks were diluted to a working concentration of 2E+12 genome copies (gc) per milliliter (gc/mL). Individual mice were injected with 1E+11 gc of AAV8-shNS1 or AAV8-shScr (50 μL) via intrasplenic or portal vein injection. Two weeks after the injection, liver tissues were collected for RNA isolation, 1^st-strand cDNA synthesis, and qPCR analysis. The results showed...

Discussion

Gene KD by viral delivery of either a shRNA-expressing construct in a wildtype background^24,25 or Cre recombinase-expressing construct in a floxed background²⁶ is a powerful way to interrogate gene function in vivo in an inducible, time-controlled manner. An ideal delivery method for in vivo gene KO/KD studies should achieve a high KO/KD efficiency in the organ of interest, and, in addition, the procedure itself should be...

Materials

Name	Company	Catalog Number	Comments
Amicon filter centrifugation units	MilliporeSigma	UFC9100	Fast ultrafiltration
AV5-siRNA-GFP	Addgene	#124972	Plasmid
Competent DH5α bacteria	Invitrogen	#18265-017	Chemically competent strain for cloning
HpaI	New England Biolabs	R0105	Restriction enzyme
iMFectin transfection reagent	GenDepot	I7100-101
M-MLV reverse transcriptase	Promega	M1708	RNA-dependent DNA polymerase
MyiQ single-color real-time PCR detection system	Bio-Rad	BUN9740RAD	qRT-PCR
Omega endotoxin-free kit	Bioteck	D6915-03	Plasmid DNA midi prep
Random hexamers	Invitrogen	48190-011
Supermix SYBR green reagent	Bio-Rad	1708882	Real-time PCR applications
T4 DNA Ligase	Promega	M1801
TRIzol Reagent	Life Technologies	15596-018	Isolation of high-quality total RNA
XhoI	New England Biolabs	R0146	Restriction enzyme