Intrabody gene therapy ameliorates motor, cognitive, and neuropathological symptoms in multiple mouse models of Huntington's disease

Abstract

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease resulting from the expansion of a glutamine repeat in the huntingtin (Htt) protein. Current therapies are directed at managing symptoms such as chorea and psychiatric disturbances. In an effort to develop a therapy directed at disease prevention we investigated the utility of highly specific, anti-Htt intracellular antibodies (intrabodies). We previously showed that V(L)12.3, an intrabody recognizing the N terminus of Htt, and Happ1, an intrabody recognizing the proline-rich domain of Htt, both reduce mHtt-induced toxicity and aggregation in cell culture and brain slice models of HD. Due to the different mechanisms of action of these two intrabodies, we then tested both in the brains of five mouse models of HD using a chimeric adeno-associated virus 2/1 (AAV2/1) vector with a modified CMV enhancer/chicken beta-actin promoter. V(L)12.3 treatment, while beneficial in a lentiviral model of HD, has no effect on the YAC128 HD model and actually increases severity of phenotype and mortality in the R6/2 HD model. In contrast, Happ1 treatment confers significant beneficial effects in a variety of assays of motor and cognitive deficits. Happ1 also strongly ameliorates the neuropathology found in the lentiviral, R6/2, N171-82Q, YAC128, and BACHD models of HD. Moreover, Happ1 significantly prolongs the life span of N171-82Q mice. These results indicate that increasing the turnover of mHtt using AAV-Happ1 gene therapy represents a highly specific and effective treatment in diverse mouse models of HD.

Figure 1.

Happ1 treatment improves rotarod performance in four HD mouse models. Mice were tested on an accelerating rotarod for a maximum of 300 s. A, Male R6/2 and wt littermates were tested weekly: Happ1 treatment significantly improves performance during weeks 9–12, while V_L12.3 treatment degrades performance during weeks 10–12. B, Male N171-82Q and wt littermates were tested every other week. Happ1 treatment improves performance to a level not different from wt animals from 22 weeks until 38 weeks. C, Male YAC128 and wt littermates were tested monthly and Happ1 (but not V_L12.3) treatment significantly improves performance at months 3, 4, and 7. D, YAC128 rotarod performance only for GFP- and Happ1-treated mutant groups is shown for simplicity. E, Male and female BACHD mice were tested monthly: Happ1 treatment significantly improves performance at months 5 and 6. Asterisks indicate difference between GFP- and iAb-treated mutants. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

Happ1 treatment improves beam-crossing performance in four HD mouse models. Time to cross the center 80 cm of a square, 1-m-long, 6-mm-wide beam was measured. A, Male R6/2 and wt littermates were tested weekly: Happ1 treatment improves performance during weeks 9–11, while V_L12.3 treatment degrades performance at 9 and 10 weeks. B, Male N171-82Q and wt littermates were tested every other week: Happ1 treatment improves performance at 18 weeks of age and older. C, Male YAC128 and wt littermates were tested monthly: Happ1 treatment improves performance at 3 and 4 months. D, Male and female BACHD mice were tested monthly: Happ1 treatment improves performance at 6 months. Asterisks indicate difference between GFP- and iAb-treated mutants. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

Happ1 treatment improves climbing and prevents clasping in HD transgenic mice, and V_L12.3 or Happ1 treatment prevents the amphetamine-induced rotation phenotype caused by mHDx-1 lentivirus. A, B, Mice were placed at the bottom of a vertical wire mesh tube and observed for 5 min. The time when all four feet were off the ground was scored as climbing time. A, Seven-month-old GFP- and V_L12.3-treated YAC128 mice have impaired climbing compared with wt mice. This is rescued by Happ1 treatment. B, Happ1 treatment improves climbing time in 6-month-old BACHD mice. C, Twenty-week-old male N171-82Q and wt littermates were suspended by the tail, observed for 1 min, and given a clasping score as follows: no clasping = 0, forelimb clasping = 1, hindlimb clasping = 2. D, Wild-type mice were injected unilaterally at 4 weeks of age with mHDx-1 or GFP lentivirus plus GFP-, V_L12.3-, or Happ1-AAV. At 10 weeks of age mice were assayed for amphetamine-induced rotation. Uninjected (naive) animals were tested as a negative control. Animals injected with mHDx-1 lentivirus and GFP-AAV exhibit many ipsilateral rotations in response to amphetamine. V_L12.3 or Happ1 prevents this phenotype. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

Happ1 treatment normalizes open field behavior in full-length transgenic models of HD. Mice were observed for 10 min during exploration of an open field. Anxiety was inferred by scoring entries into, and time spent in, the center of the open field. A, C, There was a trend toward increased center entries for both models in response to Happ1 treatment, but it was not significant. B, D, Happ1 treatment increases time spent in the center of the open field in 7-month-old YAC128 mice (B) and 6-month-old BACHD mice (D). *p < 0.05, **p < 0.01.

Figure 5.

Happ1 treatment ameliorates the learning deficit of YAC128 mice. A, To assay for preference of a known object in a novel location, mice were habituated to an open field for 10 min. After a 5 min ITI, they were exposed for 5 min to novel objects in the upper corners of the open field (T1). Investigation of the novel objects was scored. After another 5 min ITI, the mice were reintroduced to the same field with the object previously in the upper right corner moved to the lower right corner (T2) for 5 min. The percentage of the investigations of the target object (the one in the new location) was scored. A score of 50% would indicate no preference. B, On the next day mice were tested for preference for a novel object. Mice were rehabituated to the open field for 10 min. After a 5 min ITI, they were exposed for 5 min to two objects in the upper corners of the open field (T1). Investigation of the objects was scored. After another 5 min ITI the mice were reintroduced to the same field with the object in the upper right corner replaced with a completely novel object in the same location. The percentage of the investigations of the target object (the completely novel one) was scored. A score of 50% would indicate no preference. C, D, Seven-month-old YAC128 and wt littermates were tested. Wild-type mice display a preference for the novel object location (C) and a trend toward a preference for the novel object (D). This does not reach significance, but when data from the three wt treatment groups are pooled, the preference is significant (p < 0.01). GFP- and V_L12.3-treated YAC128 mice show no preference for either object in either paradigm, indicating a learning deficit. Happ1 treatment improves this deficit. E, F, GFP- and Happ1-treated BACHD mice show no preference for either object in either paradigm. There is a trend toward a preference for the novel object in Happ1-treated BACHD mice, but it does not reach significance. *p < 0.05, ***p < 0.001.

Figure 6.

mHDx-1 lentivirus causes neuron-specific toxicity in the striatum, which is reduced by V_L12.3 or Happ1 treatment. Mice were injected unilaterally with mHDx-1 or GFP lentivirus plus GFP-, V_L12.3-, or Happ1-AAV. Areas with loss of DARPP-32 staining were analyzed. A, Areas of DARPP-32 loss (bottom) also show loss of NeuN-positive cells, indicating death of neurons in these areas. B, Topro-3 iodide nuclear stain shows the presence of cells in lesioned areas, indicating that toxicity is neuron specific. C, Areas of DARPP-32 loss show increased GFAP staining, indicating increased inflammation in lesioned areas. In A–C, the top shows GFP lentivirus and the bottom shows mHDx-1 lentivirus. D–F, Coinjection of either Happ1 or V_L12.3 with mHDx-1 reduces the area and intensity of DARPP-32 loss. D, Adjacent coronal sections stained either for mHDx-1 (green) or DARPP-32 (red). DARPP-32 loss is reduced in the presence of either intrabody. E, The ratio of the area of total DARPP-32 loss to the transduced area was compared to assess lesion size. Lesions are significantly smaller in the presence of either intrabody. Three sections per mouse were analyzed. F, The ratio of DARPP-32 staining fluorescence intensity in the transduced area of the striatum to DARPP-32 staining fluorescence intensity in the same size area of the uninjected striatum was compared to assess the severity of the lesion. Lesions are less severe in the presence of iAb. Three sections per mouse were analyzed. *p < 0.05, **p < 0.01, Scale bars, 50 μm.

Figure 7.

V_L12.3 or Happ1 decreases Htt aggregation in the lentiviral and R6/2 HD models. A–C, Three sections each of 6-weeks-postsurgery mHDx-1 lentivirus-injected (A) and 10-week-old (B, C) R6/2 brains were stained for Htt. A, The ratio of striatal aggregates to transduced area is reduced by both intrabodies. B, The number of small Htt aggregates per 250 μm² is reduced by both intrabodies. C, Happ1 treatment reduces the number of intranuclear inclusions per 250 μm². *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8.

Happ1 treatment reduces ventricular enlargement in three HD mouse models. A–D, Ventricle area was measured at approximately bregma 0.0 mm in three sections each from 10-week-old male R6/2 and wt littermates (A, B), 7-month-old male YAC128 and wt littermates (C), and 6-month-old male and female BACHD mice (D). Both R6/2 and YAC128 mice display increased ventricle size compared with wt littermates. B–D, Happ1 treatment reduces ventricular enlargement in R6/2 mice (B), YAC128 mice (C), and BACHD mice (D). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 9.

Happ1 treatment improves body weight and survival of N171-82Q mice. A, R6/2 mice weigh significantly less than wt mice from 10 weeks of age until death, and there is no effect of iAb treatment. Asterisks indicate a difference between GFP-treated wt and R6/2 mice. B, Happ1-treated N171-82Q mice weigh less than wt littermates, but they also weigh more than GFP-treated mutants. Asterisks indicate a difference between GFP- and Happ1-treated N171-82Q mice. C, While YAC128 mice trend toward weighing more than wt mice, there is no effect of iAb treatment. D, While male BACHD mice weigh more than female mice, there is no effect of iAb treatment. Asterisks indicate a difference between GFP-treated male and female mice. E, Happ1 treatment has no effect, while V_L12.3 decreases survival of R6/2 mice. F, Happ1 treatment increases maximum survival of N171-82Q mice by 33% from 30 weeks to 40 weeks of age. *p < 0.05, **p < 0.01, ***p < 0.001.