HT1 = hereditary tyrosinemia type-1
HPD = 4-hydroxyphenylpyruvate dioxygenase
upstream enzyme of HT1
this is what NTBC targets and inhibits
NTBC = 2-( 2-nitro-4-trifluoromethylbenzoyl )−1,3-cyclohexanedione
FAH = fumarylacetoacetate hydrolase
Porcine = Species : Sus Scrofa = "domesticated pig"
AFP = alpha-fetoprotein
HCC = hepatocellular carcinoma
LV = lentiviral
AAT = liver-secific alpha-1-antitrypsin promotor
VCG = vector copy per genome
HCR-AAT = hepatic control region enhancer and alpha-1 antitrypsin promotor
HT1 :
If caught before 1 month of age , NTBC administration can usually convert HT1 into HT3 , a more benign phenotype and prevent HCC.
Still unanswered if NTBC can continue to prevent HCC for longer than 30 years
NTBC Problems :
requires daily administration
requires bloodwork every 3-6 months to check AFP levels
can cause elevated tyrosine concentration
Authors previously found ex vivo LV gene transfer followed by autologous transplantation of corrected hepatocytes cured mouse and pig models of HT1.
Autologous transplantation requires removing part of the liver to avoid an immune rejection
In vivo gene therapy advantages :
Previous in vivo HT1 gene editing attempts in mice :
Gene editing in larger animal models hasn't been tried
The mouse model for HT1 is very different than the human and pig phenotypes :
Problems with other gene editing techniques :
AAV :
Cas9 :
Episomal vectors create transgene expression that gets diluted every time the cell replicates
Benefits of HIV-derived LV Vectors :
HIV infection in humans is rare
LV can house a large gene cassette
Success has been seen in large animal models of hemophilia B
Cantore et al. showed that intraportal administration of a LV vector carrying the cFIX transgene is safe in dogs
The authors evaluated both the effectiveness and safety profile of in vivo liver-directed LV gene therapy for the treatment of a pro-cancerous metabolic disease, HT1
They tested deliver LV into an ear vein and into the portal vein in the liver.
Both locations showed these inflammatory cytokines peaking at 1 hour post injection , and then decreasing back to normal after 6 to 12 hours
Before the experiments began , they administered immunosuppressants
However , some allergic reactions and hypertension were observed.
A vasopressor ( epinephrine IV ) was administered
They then used percutaneous ultrasound-guided portal vein administration of the LV vector carrying the human FAH transgene in four FAH−/− pigs
Table 1 displays the dosing protocol used on 6 different pigs
No. 169 ( Carmen ) died 2 days after the treatment
autopsy showed severe hypertrophic cardiomyopathy
Hypertrophic cardiomyopathy is a known complication in Landrace pigs
They stopped administering NTBC when they started infusing the vector.
They continued to cycle NTBC on and off until they were able to maintain a healthy weight
Pig No. 166 achieved NTBC-independent growth at day 98 post treatment and Pig No. 167 at day 78 post-treatment, both after four cycles on the drug (Fig. 2a).
At the time of planned euthanasia (1 year old, 337 days post-treatment), these pigs had been off NTBC for 239 and 259 days respectively with no health issues, and with and average weight gain of between 0.6 and 0.9 kg per day, which is normal for healthy, wild-type Large White and Landrace pigs.
Tyrosine levels and liver function tests were performed
At 142 days post-treatment the levels became comparable to wild-type pigs
tyrosine was at 79 µM in No. 166 and 63 µM in No. 167
Aspartate aminotransferase (AST), alkaline phosphatase (ALP), ammonia, and bilirubin levels all became normal as well
Liver Function Tests also came back as normal
ALP in both animals and gamma-glutamyl transferase (GGT) in one animal were elevated only once during the experiment
They elected to euthanize Pig No. 162 , 60 days after LV treatment
They harvested the liver and took around 100 left , middle , and right lobe sections
They found an even distribution of clonally expanded FAH-positive hepatocytes ,
No active signs of inflammation , fibrosis , or necrosis
In contrast, no FAH-positive cells or liver abnormalities were found in Pig No. 169 48 h post-treatment
In the longer-term animals ( Pigs 166 and 167 ) :
225 days post-treatment : 2nd and 3rd row :
they performed laparoscopic liver biopsies from the left and right liver lobes
extensive liver repopulation of FAH-positive hepatocytes
multiple abnormalities were seen in 50 to 80% of the liver of both animals :
337 days post-treatment : 4th and 5th row :
Untreated or chronically undertreated FAH−/− adult pigs (No. 266, prior experiment, Fig. 2a) develop cirrhosis, adenomas, and ultimately HCC at 12 months, as shown here with hepatocellular morphological derangement, extensive stage 4 fibrosis, and significant AFP staining (Fig. 3c, right column).
In the LV-FAH treated animals, no such lesions were present grossly after sectioning the entire organ into one centimeter slices, and none of these pathological features were present in any sections evaluated histologically.
Untreated Pig No. 266 showed appropriately elevated AFP at birth similar to Nos. 166 and 167 (Fig. 2c) and to wild-type controls; however, at the time of necropsy (1 year old) No. 266 showed increased AFP levels to 168 ng/mL, further supporting the development of HCC in this animal as compared to Nos. 166 and 167, whose AFP levels at the time of necropsy are below 0.5 ng/mL
Ki-67 and TUNEL staining were performed to assess levels of hepatocyte proliferation and apoptosis
While Ki-67 staining expectedly increased during the process of liver repopulation with FAH-positive hepatocytes, it was at wild-type levels by 337 days post-treatment, mirroring levels of inflammation and fibrosis seen on routine histology.
The performed PCR analysis on 18 different tissue types to see if FAH was present in any off target locations.
When delivered via the portal vein :
When delivered via systemic ear injection :
They next characterized the in vivo LV-FAH vector integration profile in hepatocytes
Next-generation sequencing and bioinformatics analysis was performed : ( Fig. 4b-h )
Integration sites were present across the genome in all animals
No significant differences in general LV integration profile in the liver between the two long-term animals
However, integration profile in the 60-day animal ( No. 162 ) showed some unique characteristics :
No. 162's relative integration frequency was higher in exons as compared to introns
@ 337 days = Nos. 166 and 167's integration was more frequent in introns than in exons
Relative LV integration frequency remained similar across gene expression levels
At both time points ( 60 and 337 days ) , LV integration was very rare in CpG-rich promoter regions relative to their percentage of the genome as compared to non-CpG islands. ( Fig. 4e )
with a trend toward non-tumor coding genes for the longer-term animals ( Fig. 4f )
Integration was most prevalent downstream of transcription start sites
Table 2 = The top ten most common genes in terms of integration frequency for each animal and tissue type
Gene Set Enrichment Analysis (GSEA) demonstrated enrichment of the PI3K-Akt and TGFb signaling pathways only in No. 167, while No. 166 demonstrated no hits
There were no changes in gene expression in any tumor-related genes
RNAseq was done to demonstrated expression profile similarities between wild-type, LV-FAH treated, and NTBC-treated pigs as compared to tumor-positive pig liver
LV-FAH treated animals at 6 months showed an expression profile similar to both FAH−/− sick or untreated animals and NTBC-sustained FAH−/− animals
@ 12 months post-treatment where liver repopulation with healthy ,
Gene expression was compared to 6 month and 12-month time points for LV-FAH treated animals
They found gene expression differences clustered into disease pathways of importance in the progression of tyrosinemia,
Finally , they performed a principal component analysis including all genes (n = 1260) from quartile 4
i.e., genes that met our filtering criteria with the highest overall level of expression when divided into quartiles
Percutaneous portal vein infusion of LV-FAH was well tolerated, with no systemic inflammatory reaction.
When the same vector was delivered systemically, however, animals developed an acute hypotensive anaphylactoid-like reaction requiring vasopressor support
likely related to a transient rise in circulating cytokines TNF-α, IL-6, IL-8 and INF after exposure to LV particles
Mice exposed to LV particles are known to generate similar cytokine responses
Percutaneous portal vein cannulation is an advanced but obtainable skill that would allow for liver-directed gene therapy in humans
Percutaneous portal vein delivery is better at targeting the liver than systemic ear vein delivery
Initial lentiviral integration into the liver was measured at less than one VCG
well below FDA recommendations of five VCG
This number rapidly increased to ten in our long-term animals
because corrected hepatocytes expanded to repopulate the liver
In vivo LV gene therapy cured HT1 in a more rapid timeframe than ex vivo gene therapy using the same vector.
Two long-term pigs demonstrated NTBC independence at day 78–98 post-treatment
By comparison , the authors previous ex vivo study resulted in NTBC independence at 93–150 days post-treatment
All liver function tests in experimental animals were found to be at wild-type levels at four and 5 months post-treatment
By day 337 , liver histology in the treated animals mirrored the wild-type.
There was no evidence of chronic liver damage or increased AFP staining in our experimental animals
even after 8 months off the protective drug NTBC
This is especially important because we have also demonstrated here that FAH−/− animals that do not receive gene therapy and are chronically undertreated with NTBC
develop extensive fibrosis within weeks and can progress to hepatic adenomas within 6 months of life and HCC prior to a year of life
HT1 pigs develop HCC in a fibrosis-dependent manner like humans and unlike mice
Previous studies using a non-lentiviral retrovirus (GlFSvNa) in mice to treat HT1 in vivo showed metabolic correction of the phenotype but increased rates of HCC
The assumption was made that the increased HCC rates were due to a number of uncorrected hepatocytes that persisted and then progressed to HCC
This safety concern has precluded gene therapy development for HT1 for more than two decades
Mice develop adenomas and HCC through a different set of molecular derangements when compared to humans and pigs
pig model of HCC has been shown to develop HCC through a similar set of molecular derangements as seen in most human HCCs. Including :
In humans, including HT1 patients, HCC develops in the setting of an extensively inflamed, fibrotic, or cirrhotic liver.
The tissue setting in which HCC develops is markedly different in humans and rodents
chronic hepatitis and cirrhosis are uncommon lesions in the livers of rats and mice that develop HCC
Therefore, mice develop adenomas and HCC in the liver through an inflammatory and fibrotic-independent process
where in humans and pigs, especially in the setting of HT1
At the end of the therapy , they found no evidence of any chronic inflammatory changes anywhere in the liver
They didn't find any evidence of adenomas or HCC in either of our long-term experimental animals.
They also didn't find enrichment of any tumor-related pathways on GSEA
The experiment animals were cycled on and completely off NTBC
The LV-FAH vector showed a benign integration profile in pig hepatocytes after in vivo delivery
Evaluation of 17,000–39,000 unique integration sites in all animals revealed no genotoxic events
Lentivirus tends to integrate into gene coding regions without an obvious preference for transcription start sites, promoter regions or other regulatory sites of the genome
This study , along with their previous research , establishes a favorable safety profile
In this study , they observed integration bias in liver DNA
They found LV-FAH integration was increased at the FAH and SERPINA1 loci
Motivation :
Gene expression associated with inflammation, fibrosis, and HCC in the liver has been well characterized.
RNA-seq analysis shows bringing back FAH expression should return to wild-type after 12 months of treatment
Pigs treated with LV-FAH have normal, undetectable levels of AFP at 1 year
The authors proposed LV-FAH is an acceptable advancement in the treatment of HT1 in lieu of chronic NTBC maintenance therapy
which does not prevent development of inflammation, fibrosis, and HCC
Furthermore, FAH-deficient patients optimally treated with NTBC are relegated at best to suffer from the HT3 phenotype, which can be debilitating over time.
HT1 patients are still in need of a true cure, such as that offered by LV-FAH.
FAH-positive hepatocytes have a "characteristic expansion".
Result :
Limitations :
small number of animals treated and analyzed
Dosages were based off of previous in vivo LV large animal studies and from their own research in mice.
experimental pig livers did not undergo CT or MRI imaging, but rather gross histological examination after 0.5 cm sectioning
The PCR screening in different tissues doesn't distinguish between cell types
Pigs were also not surveilled for HCC development across their entire lifespan
Further Research :
Conclusions :
They designed a LV vector containing the human FAH gene
They used a liver-specific promotor ( HCR-AAT )
currently being trialed in humans for the treatment of hemophilia B
In order to generate viral vectors ,
the LV-SFFV-eGFP or LV-AAT-huFAH expression construct ,
together with the packaging plasmid p8.91 and the vesicular stomatitis virus glycoprotein G-encoding plasmid pVSV-G ,
was transfected into 293 T/17 cells (CRL-11268, ATCC, Manassas, VA) using 1 mg/ml polyethylenimine
polyethylenimine = transfection agent , condenses DNA into positively charged particles
Lenti-X Provirus Quantitation Kit (Clontech, Mountain View, CA).
Pigs were pre-treated according to a previously established immunosuppression protocol
Ear vein injection of LV-FAH was performed in one 6-week-old FAH−/− pig, at a dose of ~
Because systemic LV-FAH administration led to a systemic inflammatory response syndrome (SIRS)-type response in this animal ,
Genomic DNA was isolated from snap-frozen tissue fragments using a Gentra Puregene Tissue Kit (Qiagen, Hilden, Germany).
Representative samples were taken from sixteen distinct areas of the liver and pooled for analysis.
Three pairs of primers were designed to amplify integrated vectors in the pig genome ,
For LV-GFP, these primers were :
For LV-FAH, these primers were: